EA020578B1 - Benzimidazoles and related analogs as sirtuin modulators - Google Patents

Abstract

Provided herein are sirtuin-modulating compounds of formula (II). The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders that would benefit from increased mitochondrial activity. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

Description

This application claims the benefit of provisional application US No. 61/133938, filed July 3, 2008, the contents of which are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The family of silent informational regulatory (8IIe1i1i1aaaaaaaaaaaaaaaaaaaaaaaa) genes is a highly conservative group of genes that are present in the genomes of organisms from arbaeobacterium to higher eukaryotes. The encoded 8ΙΚ proteins are involved in various processes from the regulation of gene silencing to DNA repair. Proteins encoded by members of the 8ΙΚ gene family demonstrate high sequence conservation in the 250 amino acid central core domain. A well-characterized gene in this family is 8. segeu181ae 8ΙΚ2, which is involved in the silencing of NM loci that contain information that determines the type of mating of yeast, effects of telomere position and cell aging. Protein 8ιγ2 yeast belongs to the family of histone deacetylases. The homologue 8p2, CoBB, in 8a1top11a TurYtigst acts as ΝΑΌ (nicotinamide adenine dinucleotide) -dependent ΑΌΡ-ribosyltransferase.

The 8ιγ2 protein is a class III deacetylase that uses ΝΑΌ as a sub-substrate. Unlike other deacetylases, many of which are involved in gene silencing, 8ιγ2 is not sensitive to histone deacetylase inhibitors class I and II, similar to trichostatin A (T8A).

Deacetylation of acetyl-lysine by means of 8p2 is closely related to ΝΑΌ hydrolysis, with the formation of nicotinamide and a new acetyl-ΑΌΡ ribose compound. The ΝΑΌ-dependent deacetylase activity of 8ιγ2 is essential for its functions, which may link its biological role with cellular metabolism in yeast. The homologues of 8ιγ2 mammals have a ΝΑΌ-dependent histone deacetylase activity.

Biochemical studies have shown that 8ιγ2 can easily deacetylate the amino-terminal processes of histones H3 and H4, leading to the formation of 1-O-acetyl-Α ^ Ρ-ribose and nicotinamide. Strains with additional copies of 8GK2 demonstrate increased silencing gDNA and 30% longer lifespan. It has recently been shown that additional copies of the 8GK2 homologue C. e1e§ai8, 81G-2.1, and 88i2 gene Ό. Sheliao-e81 strongly increase the life expectancy in these organisms. This suggests that the 8GK2-dependent regulatory pathway for aging arose early as a result of evolution and was well preserved. Currently, it is believed that the development of 8ιγ2 genes has occurred to enhance the body's health and resistance to stress in order to increase its survival under adverse conditions.

In humans, there are seven 8n2-like genes (8IΚT1-8IΚT5) that share a conserved catalytic domain 8ιγ2 for them. 8GCT1 is a nuclear protein with the highest degree of sequence similarity to 8p2. 8GKT1 regulates several cellular targets by deacetylation, including the p53 tumor suppressor, ΝΡ-κΒ cell signaling factor, and PHO transcription factor.

8GCT3 is a homologue of 8GCT1, which is conserved in prokaryotes and eukaryotes. The 8ΣΚ.Τ3 protein targets mitochondrial cristae through a unique domain located at the Ν-terminus. 8ΣΚ.Τ3 has ΝΑ ^ + - dependent protein deacetylase activity and is widely expressed, in particular, in metabolically active tissues. It is believed that 8ΣΚ.Τ3, when transferred to mitochondria, should be cleaved to a finer active form under the influence of mitochondrial matrix processing peptidase (MPP).

Calorie restriction has been known for over 70 years as contributing to better health and longer mammalian life. The life span of yeast, as in multicellular animals, is also prolonged as a result of interventions that resemble calorie restriction, such as low glucose. The discovery that yeast and flies that lack the 8GK2 gene does not last long with calorie restriction suggests that 8GK2 genes mediate the health-friendly effects of a calorie-restricted diet. Moreover, mutations that reduce the activity of the glucose-sensitive cAMP (adenosine 3 ′, 5′-monophosphate) -dependent (ΡΚΑ) pathway in yeast increase the lifespan in wild-type cells, but not in mutant 81g2 strains, indicating that 8GK2 is probably a key component that works along the path of calorie restriction.

SUMMARY OF THE INVENTION

This application presents new sirtuin-modulating compounds and methods for their use.

In one aspect, the present invention provides sirtuin-modulating compounds of structural formulas (I) and (II), as described in detail below.

In another aspect, the present invention provides methods for using sirtuin modulating compounds or compositions comprising sirtuin modulating compounds. In some embodiments, sirtuin-modulating compounds that increase the level and / or activity of sirtuin protein can be used for various therapeutic applications, including the treatment and / or prophylaxis of many different diseases and disorders, including, for example, treatment

- 1,020,578 of a subject suffering from or susceptible to insulin resistance, metabolic syndrome, diabetes or its complications, or increased insulin sensitivity in the subject.

Sirtuin-modulating compounds can be entered separately or in combination with other compounds, including other sirtuin-modulating compounds or other therapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

As used in this application, the following terms and phrases should have the meanings indicated below. Unless otherwise indicated, all technical and scientific terms used in this application have generally accepted meanings known to those of ordinary skill in the art.

The term agent is used in this application to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or part thereof, for example, a peptide) or an extract obtained from biological substances, such as cells or tissues of bacteria, plants , fungi or animals (in particular mammals). The activity of such agents can make them suitable as a therapeutic agent, which is a biologically, physiologically or pharmacologically active substance (or substances) that acts locally or systemically in the body of the subject.

The term bioavailable, when referring to a compound, is generally accepted in the art and refers to a form of the compound which, for this compound or part of the administered amount of this compound, allows absorption, inclusion, or makes it otherwise physiologically accessible to a subject or patient to whom it is administered.

The biologically active portion of sirtuin refers to a portion of the sirtuin protein having biological activity, such as the ability to carry out deacetylation. The biologically active parts of sirtuin may include a domain representing the core of sirtuins. The biologically active parts 8ΙΚΤ1 with access number OepVapk No. ΝΡ_036370, which cover the ΝΑΌ + binding domain and substrate-binding domain, for example, may include, without limitation, amino acids 62-293 with access number OepVapk No. ΝΡ_036370, which are encoded by nucleotides 237932 with access number OepVapk No. ΝΜ_012238. Therefore, this area is sometimes indicated as representing the core domain. Other biologically active parts 8ΙΚΤ1, also sometimes referred to as core domains, include the amino acid region 261-447 with access number OepWapc No. ΝΡ_036370, which are encoded by nucleotides 834-1394 with access number OepWapk No. ΝΜ1_12238; the amino acid region 242-493 with the access number OepWapc ΝΡ_036370, which are encoded by nucleotides 777-1532 with the access number OepWapk No. ΝΜ_012238; or the amino acid region 254-495 with access number OepWapk No. ΝΡ_036370, which are encoded by nucleotides 813-1538 with access number OepWapk No. ΝΜ_012238.

The term pets refers to cats and dogs. As used in this application, the term dog (dogs) refers to any member of the Sash GatShagT species. which includes a large number of different breeds. The term cat (s) refers to a feline animal, including domestic cats and other members of the ReChay family, of the ReY8 genus.

Diabetes refers to high blood sugar or ketoacidosis, as well as chronic general metabolic disorders resulting from a prolonged condition characterized by high blood sugar, or as a result of reduced glucose tolerance. Diabetes covers both forms of the disease, type I and type II (insulin-independent diabetes mellitus or ΝΙΌΌΜ). With regard to diabetes, risk factors include the following: waist size greater than 40 inches (101.6 cm) for men or 35 inches (88.9 cm) for women, blood pressure 130/85 mm Hg or higher, triglycerides above 150 mg / dl, fasting blood glucose greater than 100 mg / dl, or high density lipoproteins less than 40 mg / dl in men or 50 mg / dl in women.

The term ΕΌ ₅₀ is generally accepted in the art. In some embodiments, ΕΌ ₅₀ means a dose of a drug that provides 50% of the maximum response or effect, or, alternatively, a dose that provides a predefined response for 50% of the test subjects or drugs. The term BO _5o is generally accepted in the art. In some embodiments, LO ₅₀ means a dose of a drug that is lethal to 50% of the test subjects. The term therapeutic index is a term generally accepted in the art that refers to the therapeutic index of a drug, defined as ΡΌ ₅₀ / ΕΌ ₅₀ .

The term hyperinsulinemia refers to the condition of an individual in which the level of insulin in the blood is higher than normal.

The term insulin resistance refers to a condition in which a normal amount of insulin causes an insufficient biological response compared to a biological response in a subject who does not have insulin resistance.

The insulin resistance disorder, as discussed herein, refers to any disease or condition that is caused by insulin resistance or contributes to insulin resistance. Examples include diabetes, obesity, metabolic syndrome, insulin resistance syndromes, Syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease, including stroke, coronary artery disease or heart attack myocardium, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including coronary heart disease levitation, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, some types of cancer (such as endometrial, breast, prostate, colon) pregnancy complications, poor female reproductive health (such as a violation of the mental cycle, infertility, irregular ovulation, polycystic ovary syndrome (PCO8)), lipodystrophy, cholesterol-related disorders such as bile calculi, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis and the prevention and treatment of bone loss, such as osteoporosis.

The term livestock refers to domesticated four-legged animals, which include animals that are raised for meat and various by-products, such as cows, including cattle and other members of the Voe family, pigs, including domestic pig and other members of the genus §iv, sheep, including a sheep and other members of the genus Ονίβ, domestic goats and other members of the genus Sarga; to domesticated four-legged animals that are raised for special purposes, for example, for use as pack animals, such as horses, including domestic horses and other members of the Eszibas family. clan Estschv.

The term mammal is known in the art, and examples of mammals include humans, primates, livestock (including cows, pigs, etc.), domestic animals (e.g. dogs, cats, etc.) and rodents (e.g. mice and rats).

Obese or obese individuals are generally individuals having a body mass index (ΒΜΙ) of at least 25 or more. Obesity may or may not be associated with insulin resistance.

The terms parenteral administration and administered parenterally are generally accepted in the art and refer to methods of administration other than enteral and local administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

A patient, subject, individual, or host refers to either a human or animal other than a human.

The term pharmaceutically acceptable carrier is generally accepted in the art and refers to a pharmaceutically acceptable substance, composition or carrier, such as a liquid or solid excipient, diluent, excipient, solvent or encapsulating substance, involved in the transfer or transportation of any composition or component of interest . Each carrier must be acceptable in the sense that it must be compatible with the composition of interest and its components and not be harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffered solutions; and (21) other non-toxic compatible substances used in pharmaceutical compositions.

The term prophylactic or therapeutic treatment is generally accepted in the art and refers to the administration of a drug to a host. If it is administered before the clinical manifestation of an undesirable condition (for example, a disease or other undesirable condition of the host animal), then the treatment is prophylactic, i.e. protects the host against the development of an undesirable state, whereas if it is administered after the manifestation of an undesirable state, the treatment is therapeutic (i.e., it is intended to reduce, alleviate or maintain an existing undesirable state or side effects caused by it).

- 3,020,578

The term pyrogen-free, when referring to a composition, refers to a composition that does not contain pyrogen in an amount that could lead to an adverse effect (e.g., irritation, fever, inflammation, diarrhea, respiratory distress syndrome, endotoxic shock, etc.) in a subject to whom such a composition has been administered. For example, the term is intended to encompass compositions that do not or essentially do not contain endotoxin, such as, for example, a lipopolysaccharide (L8).

Replicative cell life refers to the number of daughter cells produced by a single mother cell. Chronological aging or chronological life expectancy, on the other hand, refers to the period of time during which a population of non-dividing cells remains viable, being devoid of nutrients. An increase in cell life or extension of cell life, when applied to cells or organisms, refers to an increase in the number of daughter cells produced by one cell; increase the ability of cells or organisms to cope with stress and deal with damage, for example, DNA, proteins; and / or increasing the ability of cells or organisms to survive and exist for a longer time under specific conditions, for example, stress (e.g. heat shock, osmotic stress, high-energy radiation, chemically induced stress, DNA damage, insufficient salt, insufficient nitrogen or insufficient nutrient levels). Life expectancy can be increased by at least about 20, 30, 40, 50, 60%, or in the range from 20 to 70%, from 30 to 60%; from 40 to 60% or more using the methods described in this application.

Sirtuin activating compound refers to a compound that increases the level of sirtuin protein and / or increases at least one activity of sirtuin protein. In an illustrative embodiment, the sirtuin activating compound may increase at least one biological activity of the sirtuin protein by at least about 10, 25, 50, 75, 100% or more. Examples of biological activities of sirtuin proteins include deacetylation of, for example, histones and p53; life extension; increased genomic stability; silencing transcription and control of segregation of oxidized proteins between mother and daughter cells.

The sirtuin protein belongs to a member of the sirtuin deacetylase protein family or, preferably, to the δίτ2 family, which includes yeast διτ2 (SepWap access number P53685), δίτ-2.1 C. e1edap5 (SepWap access number ΝΡ_501912) and human δΙΚΤΙ (SepBap36 access number 00_0_0_0 or AP083106)) and δΙΚΤ2 (access number SepVapk ΝΜ_012237, ΝΜ_030593, ΝΡ_036369, ΝΡ_085096 and ΑΡ083107) proteins. Other members of the family include four additional 5> H2-like. \ Yeast genes called NBT genes (two homologues δ дваτ) ΗδΤΙ, ΗδΤ2, Н8Т3 and Н8Т4 and five other human homologues ΗδΙΚΤ3, ΗδΙΚΤ4, ΙιδΙΚΤΤ ΙΚΤδΙΚΤ6 and Βδ (7 (bееb1. 1995) Cepez Όεν. 9: 2888 apD Mercury e! A1. (1999) VVKS 260: 273). Preferred sirtuins are those that are more similar to δΙΚΤΙ, i.e. ΗδΙΚΤΙ and / or διτ2 than with δΙΚΤ2, such as terms having at least a part of the кон-terminal sequence that is present in δΙΚΤΙ and absent in δΙΚΤ2, for example, as it has δΙΚΤ3.

The δΙΚΤΙ protein belongs to the member of the διτ2 family of sirtuin deacetylases. In one embodiment, the δΙΚΤΙ protein includes διτ2 yeast (SepWapk access number P53685), διτ-2.1 C. e1edap8 (SepWap access number ΝΡ_501912), human δΙΚΤΙ (SepWap access number ΝΜ_012238 or ΝΡ_036370 (or AP083106)) and their fragments . In another embodiment, the δΙΚΤΙ protein comprises a polypeptide comprising a sequence consisting of or essentially consisting of the amino acid sequence shown in SepWap, access numbers ΝΡ_036370, ΝΡ_501912, ΝΡ_085096, ΝΡ_036369 or Ρ53685. δΙΚΤΙ proteins include polypeptides comprising all or part of the amino acid sequence shown in SepWap, access numbers ΝΡ_036370, ΝΡ_501912,

ΝΡ_085096, ΝΡ_036369 or Ρ53685; the amino acid sequence presented in SepWapc, access numbers ΝΡ_036370, ΝΡ_501912, ΝΡ_085096, ΝΡ_036369 or Ρ53685, with from 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; amino acid sequence, which is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to the sequence shown in SepWap, access numbers ΝΡ_036370, ΝΡ_501912, ΝΡ_085096, ΝΡ_036369 or Ρ53685, and their functional fragments. The polypeptides of the present invention also include homologues (e.g., orthologs and paralogs), variants or fragments presented in SepVap with access numbers ΝΡ_036370, ΝΡ_501912, ΝΡ_085096, ΝΡ.®6369 or Ρ53685.

δΙΚΤ3 protein refers to a member of the sirtuindeacetylase protein family and / or to the δΙΚΤΙ protein homologue. In one embodiment, the δΙΚΤ3 protein includes human δΙΚΤ3 (SepWapk access number AAN01042, ΝΡ_036371 or ΝΡ_001017524) and murine δΙΚΤ3 (SepWapk access number ΝΡ_071878) proteins and their equivalents and fragments. In another embodiment, the δΙΚΤ3 protein comprises a polypeptide comprising a sequence consisting of or essentially consisting of the amino acid sequence shown in SepWap, access numbers AAN01042, ΝΡ_036371, ΝΡ_001017524 or ΝΡ_071878. δΙΚΤ3 proteins include polypeptides comprising all or part of the amino acid sequence presented in SepBap, access numbers AAN01042, ΝΡ_036371, ΝΡ_001017524 or ΝΡ_071878; the amino acid sequence shown in SepWap access numbers

- 4,020,578

AAN01042, ΝΡ_036371, ΝΡ_001017524 or ΝΡ_071878, s from 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; amino acid sequence, which is at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% identical to the sequence presented in OepWapk, access numbers AAN01042, ΝΡ_036371, ΝΡ_001017524 or ΝΡ_071878, and their functional fragments .

The polypeptides of the present invention also include homologues (e.g., orthologs and paralogs), variants or fragments presented in OEPWap with access numbers AAN01042, ΝΡ_036371, ΝΡ_001017524 or ΝΡ_071878. In one embodiment, an 8ΙΚΤ3 protein comprises a 8ΙΚΤ3 fragment of a protein that is formed by cleavage by mitochondrial matrix processing peptidase (MPP) and / or mitochondrial intermediate peptidase (ΜΙΡ).

The terms systemic administration, systemically administered, peripheral administration and peripherally administered are generally accepted in the art and refer to the administration of a composition of interest, therapeutic or other means, other than direct administration to the central nervous system, so that it enters the patient’s system, thus, metabolism and other similar processes.

The term therapeutic agent is generally accepted in the art and refers to any chemical particle that is a biologically, physiologically or pharmacologically active substance that acts locally or systemically in a subject. The term also means any substance intended for use in the diagnosis, treatment, relief, treatment or prevention of a disease or to enhance the desired physical or mental development and / or conditions in an animal or human.

The term therapeutic effect is generally accepted in this field and refers to a local or systemic effect in animals, in particular mammals and, more specifically, in humans, caused by a pharmacologically active substance. The phrase “therapeutically effective amount” means an amount of this substance that provides some desired local or systemic effect with a reasonable benefit / risk ratio applicable to any treatment. A therapeutically effective amount of such a substance varies depending on the subject and the condition of the disease to be treated, the body weight and age of the subject, the severity of the condition of the disease, route of administration, and similar factors, and it can easily be determined by a person skilled in the art with average skill. For example, some of the compositions described herein can be administered in sufficient quantity to produce the desired effect with a reasonable benefit / risk ratio applicable to such treatment.

Treatment for a condition or disease refers to the treatment as well as the alleviation of at least one symptom of the condition or disease.

The term visual impairment refers to a decrease in vision, which is often only partially reversible or irreversible after treatment (e.g., surgery). In particular, severe visual impairment is called blindness or loss of vision, which refers to complete loss of vision, vision worse than 20/200, which cannot be corrected with corrective lenses, or to a field of view with a diameter of less than 20 ° (radius 10 °).

2. Sirtuin Modulators

In one aspect, the present invention provides novel sirtuin-modulating compounds for treating and / or preventing a variety of different diseases and disorders, including, for example, treating a subject suffering from or sensitive to insulin resistance, metabolic syndrome, diabetes or its complications, or sensitization to insulin in a subject. The compounds disclosed in this application may be suitable for use in the pharmaceutical composition and / or one or more of the methods disclosed in this application.

In one embodiment, the sirtuin modulating compounds of the present invention are represented by structural formula (II)

or its salt, where

K ^{13 is} selected from hydrogen, halogen, -OH, -Ο ^ Ν, fluoro-substituted C1-C2 alkyl, -O- (C1-C2 fluoro-substituted alkyl), -8- (C1-C2 fluoro-substituted alkyl), C1-C4 alkyl, - (C1-C2 alkyl) -N (R ¹⁴⁾ (R ¹⁴⁾ -OSN2SN (OH) CH ₂ OH, -O- (C! -C ₄₎ alkyl, -O- _(Ci-3) alkyl-N (K ¹⁴ ) (K ¹⁴ ), -Ν (Κ ¹⁴ ) (Κ ¹⁴ ), -8- (C ₁ -C ₄ ) alkyl and C ₃ C ₇ cycloalkyl;

- 5,020,578

K is selected from hydrogen, - (C ₂ -C ₄ ) alkyl, - (C ₁ -C ₄ ) fluoro-substituted alkyl, - (C ₁ -C ₄ ) alkyl-I (K ⁷ ) (K ⁷ ), (C1-C4 ) alkyl-C (O) -Y (K ⁷ ) (K ⁷ ), - (C2-C ₄ ) alkyl-O-K ⁷ and - (C ₂ -C ₄ ) alkyl-O (K ⁷ ) -C ( O) —K ⁷ , where each K ^{7 is} independently selected from hydrogen and —C ₁ -C ₄ alkyl; or two K ⁷ bonded to the same nitrogen atom taken together with a nitrogen atom form a 4-7 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (= O) ₂ and O, where the saturated heterocycle is optionally substituted on one carbon atom —OH, —C ₁ -C ₄ alkyl, fluorine, —ΝΗ ₂ , -ΝΗ (^ - ^ alkyl), - Ν (Οι-Ο ₄ alkyl) ₂ , -ΝΗ (ΟΗ ^ Η ₂ Ο ^ ₃ ) or -Ν (ΟΗ ^ Η ₂ ΟΟΗ ₃ ) ₂ ;

K ^{4 is} selected from hydrogen, halogen, —C ^ N, fluorinated C ₁ -C ₂ alkyl, -8- (C ₁ -C ₂ ) fluorinated alkyl, C ₁ -C ₄ alkyl, -8- (C ₁ -C ₄ ) alkyl and C ₃ -C ₇ cycloalkyl;

X is selected from

-ΝΗ-σ (= ο) -ί, -с (= о) -н-т, -ин-с (= е) -1, -с (= з) ΝΗ-Ι, -ΝΗ-3 (= Ο ) -ί, -3 (= Ο) -ΝΗ-ί, -ΝΗ-5 (О) ₂ -ΝΕ ¹³ -I, -ΝΕ ¹⁵ -3 (Ο) 2-ΝΗί, -ΝΗ-С (= 0) Ο -ΐ, О-С (= 0) -ΝΗ-ί, -ΝΗ-С (= 0) ΝΗ-ί, -ΝΗ-С (= Ο) ΝΕ ¹³ -ί, ΝΕ ¹³ -0 (= 0) ΝΗ- I, -ΝΗ-ΝΕ ¹³ -I, -ΝΕ ¹³ -ΝΗ-Ι, -Ο-ΝΗ-ί, -ΝΗ-Ο-ί, -ΝΗCE ¹³ E ¹⁶ - |, -SE ¹³ E ¹⁶ -YN- |, -ΝΗ-С (= ΝΕ ¹³ > - ί, -С (= ΝΕ ¹³ ) -ΝΗ-ί, -ΝΗС (= 5) -SE ¹³ Е ¹⁶ -ί, -SE ¹³ Е ¹⁶ -С (= 3) - ΝΗ-ί, -ΝΗ-3 (O) -SK ¹³ E ¹⁶ -ί, -SE ¹³ E ¹⁶ 3 (0) -ΝΗ-ί, -ΝΗ-3 (O) 2-CE ¹³ E ^1e -ί, - CE ¹³ E ^1B -3 (Ο) ₂ -ΝΗ-ί, -ΝΗ-С (= 0) -0CE ¹³ E ¹⁶ -1, -SE ¹³ E ¹⁶ -0-С (= 0) -ΝΗ-ί, - ΝΗ-С (= 0) -ΝΕ ¹⁴ -ΟΕ ¹³ Ε ^{1 £} -ί, -ΝΗС (= 0) -ΟΕ ¹³ Ε ¹⁶ -ί and -ΟΕ ¹³ Ε ¹⁶ -ΝΗ-Ο (= Ο) -0-ί where indicates where X is associated with K ¹¹ , and

K ¹⁵ and K ¹⁶ are independently selected from hydrogen, C ₁ -C ₄ alkyl, SR ₃ and - (C ₁ -C ₄ alkyl) -CP _3;

K ¹¹ is a heterocycle that is a 4-7 membered monocyclic ring or an 8-12 membered bicyclic ring, as well as a spiro-fused and bridged polycyclic ring system comprising one or more heteroatoms selected from Ν, O and 8 atoms, with the monocyclic ring and each of the bicyclic rings independently selected from a saturated, unsaturated or aromatic ring, where K ^{11 is} optionally substituted with one or two substituents independently selected from halogen, ΡξΝ. C1-C3 alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, = 0, -O-C1-C4 alkyl, -8-K ¹⁴ , - (C1C4 alkyl) -Y (K ¹⁴ ) (K ¹⁴ ), - S (K ¹⁴ ) (K ¹⁴ ), -O-SS-Cd alkyl) -Y (K ¹⁴ ) (K ¹⁴ ), -C (O) -Y (K ¹⁴ ) (K ¹⁴ ), -C (O) -O-K ¹⁴ and - (C1-C4 alkyl) -C (O) -Y (K ¹⁴ ) (K ¹⁴ ), where each K ^{14 is} independently selected from hydrogen and-C ₁ -C ₄ alkyl; or two K ¹⁴ taken together with the nitrogen atom to which they are bonded form a 4–8 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (O) ₂ and O where, when K ¹⁴ is alkyl, such alkyl is optionally substituted with one or more groups —OH, —O— (C ₁ -C ₄ alkyl), fluoro, —ΝΗ ₂ , —NI (C ₁ -C ₄ alkyl), - S (C ₁ -C ₄ alkyl) ₂ , ΝΗ ^ Η ^ Η ^^^ or ЫС ^^ О ^ b, and when two K ¹⁴ taken together with the nitrogen atom to which they are bonded form a 4-8 membered saturated heterocycle, such a saturated heterocycle is optionally substituted at the carbon atom by the OH group, -C ₁ -C ₄ alkyl, fluoro, -ΝΗ ₂ , -ΝΗ (0ι-0 ₄ alkyl), -Y (C ₁ -C ₄ alkyl) ₂ , -UI (CH2CH ₂ OCH ₃ ) or Ν ^ Η ^ Η ^^^; and optionally substituted at any substitutable nitrogen atom with a —C ₁ -C ₄ alkyl group, a fluoro-substituted C ₁ -C ₄ alkyl, or - (CH ₂ ) ₂ —O — CH ₃ ; and

K ^{12 is} selected from a carbocycle, which is a 5-7 membered monocyclic ring system, independently selected from a saturated, unsaturated and aromatic ring system, where all ring atoms are carbon, and a heterocycle defined for K ¹¹ other than piperazine, indazole , triazole or pyrazolopyridine, wherein K ^{12 is} optionally substituted with one to two substituents independently selected from halogen, —C ^ S, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, —O — K ¹⁴ , -8- K ¹⁴ , -8 (O) K ¹⁴ , -8 (O) 2-K ¹⁴ , - (C1-C4 alkyl) -Y (K ¹⁴ ) (K ¹⁴⁾ -N (R ¹⁴⁾ (R ^14), -O _(C2-C4 alkyl) -N (R ¹⁴⁾ (R ^14), -C (O) -N (R ¹⁴⁾ (R ^14), - (C1-C4 alkyl) -C (O) -Y (K ¹⁴ ) (K ¹⁴ ), -O-phenyl, phenyl and the second heterocycle defined for K ¹¹ , and when K ¹² is phenyl, K ^{12 is} also optional substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted with 3,4-ethylenedioxy or -O- (saturated heterocycle), where any phenyl or second heterocyclic substituent of the radical K ^{12 is} optionally substituted with halogen; -ίΆΝ; C1-C ₄ alkyl, C ₁ -C ₂ fluoride-substituted alkyl group -O- (C ₁ -C ₂ fluorine-substituted alkyl), -O- (C ₁ -C ₄ alkyl) -8- (C ₁ -C ₄ alkyl ), -8 (C ₁ -C ₂ fluoro-substituted alkyl), -ΝΗ- (Οι-Ο ₄ alkyl) and -Y- (C ₁ -C ₄ alkyl) ₂ .

- 6,020,578

In some embodiments, the compound has the formula

In one embodiment of the invention, in said structures, K is hydrogen and X is

-C (= O) -ΝΗ-Ι.

In some embodiments, in the compounds of formula (II), K ^{11 is} selected from

- 7,020,578

In another embodiment, K ^{12 is} selected from

- 8,020,578

- 9,020,578

The present invention also relates to a compound of general formula (I)

or its salt, where

Э 3 1 7 3 one of Ζ and Ζ is SK, and the other of Ζ and Ζ is selected from N and SK, where

K ^{3 is} selected in each case from hydrogen, hydroxy, halogen, -ΝΝ, fluoro-substituted C1-C2 alkyl, -O- (C ₁ -C ₂ ) fluoro-substituted alkyl, -8- (C ₁ -C ₂ ) fluoro-substituted alkyl, C ₁ -C ₄ alkyl, -O- (C ₁ -C ₄ ) alkyl, -8- (C ₁ -C ₄ ) alkyl, C ₃ -C ₇ cycloalkyl;

K is selected from hydrogen, - (C ₂ -C ₄ ) alkyl, - (C ₁ -C ₄ ) fluoro-substituted alkyl, - (C ₁ -C ₄ ) alkyl-X (K ⁷ ) (K ⁷ ), (C1-C4 ) alkyl-C (O) -X (K ⁷ ) (K ⁷ ), - (C2-C ₄ ) alkyl-O-K ⁷ and - (C ₂ -C ₄ ) alkyl-X (K ⁷ ) -C ( O) —K ⁷ , where each K ^{7 is} independently selected from hydrogen and —C ₁ -C ₄ alkyl; or two K ⁷ bonded to the same nitrogen atom taken together with a nitrogen atom form a 4-7 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (= O) ₂ and O, where the saturated heterocycle is optionally substituted on one carbon atom with the group —OH, —C ₁ -C ₄ alkyl, fluorine, —ΝΗ ₂ , —ΝΗ ^ - ^ alkyl), —SCH-C alkyl) ₂ , ΝΗ ^ Η ^ Η ^^^ or ^ (CH ₂ CH ₂ OCH ₃ ) ₂ ;

K ^{1 is} selected from a carbocycle, which is a 5-7-membered monocyclic or 8-12 membered bicyclic ring system, as well as a spiro-fused and bridged polycyclic ring system, where all the ring atoms are carbon, monocyclic or each of bicyclic the rings are independently selected from a saturated, unsaturated and aromatic ring, and a heterocycle, which is a 4-7 membered monocyclic or 8-12 membered bicyclic ring system, as well as a spirocon ensirovannuyu and bridged multicyclic ring system comprising one or more heteroatoms selected from Ν, O and 8 atoms, wherein the monocyclic ring and each of the bicyclic rings are independently selected from a saturated, unsaturated or aromatic ring, where R ¹ is optionally substituted with one or two substituents independently selected from halogen (= Ν, C1-C3 alkyl, = O, C3-C7 cycloalkyl, fluoro-substituted C1-C2 alkyl, -O-K ⁸ , -8-K ⁸ , - (C1-C ₂ alkyl) ^ K ⁸ ) (K ⁸ ), -Y (K ⁸ ) (K ⁸ ), -O- (C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -O- ( C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ) and - (C1-C2 al K ⁸ ), and when K ¹ is phenyl, K ^{1 is} also optionally substituted with 3.4 fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy and a fluoro-substituted 3.4 kil) —C (O) —Y (K ⁸ ) (K ⁸ ), and when K ¹ is phenyl, K ^{1 is} also optionally substituted with 3,4 methylenedioxy, ethylenedioxy;

each K ^{8 is} independently selected from hydrogen and —C 1 -C 4 alkyl; or two K ⁸ taken together with the nitrogen atom to which they are bonded form a 4-8 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (= O) ₂ and O, where the alkyl is optionally substituted with one or more groups —OH, fluoro, —ΝΗ ₂ , ΝΗ ^ ι ^ alkyl), —SCH-C alkyl) ₂ , —CH (CH ₂ CH ₂ OCH ₃ ), or —Y (CH ₂ CH ₂ OCH ₃ ) ₂ , and the saturated heterocycle is optionally substituted at the carbon atom with the group —OH, —C ₁ -C ₄ alkyl, fluorine, —ΝΗ ^ -ΝΗ ^ - ^ alkyl), ^ CrCd alkyl) 2, NΗ (СΗ2СΗ2ОСΗ ₃ ) or -N (СΗ2СΗ2ОСΗ ₃ ) 2,

K ^{2 is} selected from a carbocycle defined for K ¹ containing at least five ring atoms and a 4-7 membered monocyclic heterocycle defined for K ¹ other than piperazine, where K ^{2 is} optionally substituted with one to two substituents independently selected from halogen, -C = Ν, C1-C3 alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, -O-K ⁸ , -8-K ⁸ , - (C1-C ₂ alkyl) -Y (K ⁸ ) ( K ⁸ ), ^ K ⁸ ) (K ⁸ ), -O- (C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -O- (C1-C2 alkyl) - S (K ⁸ ) (K ⁸ ), -C (OLC ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -C (O) -Y (K ⁸ ) (K ⁸ ), -O-phenyl, phenyl and a second heterocycle defined for K ¹ and when K ² is phenyl, K ^{2 is} also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted with 3,4-ethylenedioxy, where any phenyl or second heterocyclic K ^{2 is} optionally substituted with halogen ; -ΝΝ; C ₁ -C ₃ alkyl, C ₁ -C ₂ fluoride-substituted alkyl, -O- (C ₁ -C ₂₎ fluoride-substituted alkyl, -O- (C ₁ -C ₃₎ alkyl, 8- (C ₁ -C ₃₎ alkyl, -8- (C ₁ -C ₂ ) fluoro-substituted alkyl, -ΝΗ - ^ - ^) alkyl, and -Y- (C ₁ -C ₃ ) ₂ alkyl; and

K ^{4 is} selected from hydrogen, halogen, -ΝΝ, fluoro-substituted C ₁ -C ₂ alkyl, -8- (C ₁ -C ₂ ) fluoro-substituted alkyl, C ₁ -C ₄ alkyl, -8- (C ₁ -C ₄ ) alkyl and C ₃ -C ₇ cycloalkyl;

X is selected from

-yn-s (= o) - |, -α (= ο) -ΝΗ-ι, where it indicates where X is connected to K ¹ ; and each K ⁵ and K ^{6 is} independently selected from hydrogen, C ₁ -C ₄ alkyl, -CP ₃ and (C ₁ -C ₂ alkyl) -CP ₃ , where

- 10,020,578 when X is (O) -ΝΗ- T each Ζ ¹ and Ζ ² are CH and K ¹ is optionally substituted phenyl, then K ² is other than pyridinyl.

The compound of general formula (I) may have the following structure:

In one embodiment of the invention, in said structures, K is hydrogen. In one embodiment of the invention, in said structures X is

Examples of K ¹ include

ΛΛ / ν

- 11,020,578

In some embodiments, K ^{2 is} selected from aryl and heteroaryl. Examples of K ² include

- 12,020,578

- 13,020,578

SR ₃ .

In one embodiment of the invention, the compound of general formula (I) is selected from

or its salt.

In another embodiment of the present invention, the compound represented by structural formula II is selected from

- 14,020,578

or its salt.

Compounds of the present invention, including new compounds of the present invention, can also be used in the methods described in this application.

The compounds and their salts described herein can also form their corresponding hydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate) and solvates. Suitable solvents for the preparation of solvates and hydrates can generally be selected by one skilled in the art.

Compounds and their salts may be present in amorphous or crystalline (including cocrystalline and polymorphic) forms.

The sirtuin-modulating compounds of the present invention successfully modulate the level and / or activity of the sirtuin protein, in particular, the deacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, some sirtuin-modulating compounds of the present invention essentially do not have one or more of the following activities: inhibition of P13 kinase, inhibition of aldoreductase, inhibition of tyrosine kinase, transactivation of BORK tyrosine kinase, coronary dilation or antispasmodic activity, at concentrations of the compound that are effective for modulating the deacetylating activity of the sirtuin protein (for example, such as 8ΙΚΤ1 and / or 8ΙΚΤ3 protein).

The term heterocycle or heterocyclic means ring systems comprising one or more heteroatoms selected from, for example, Ν, O and 8 atoms, and includes 4-7 membered monocyclic and 8-12 membered bicyclic rings, as well as spiro-fused and linked bridged bonded polycyclic ring systems, where the monocyclic ring and each of the bicyclic rings are independently selected from saturated, unsaturated or aromatic rings. When the heterocycle is polycyclic, it should be understood that it is necessary that only one ring contains a heteroatom. Unless otherwise indicated, the heterocycle is optionally substituted with one or more substituent groups selected from halogen, -Ο = Ν, C ₁ -C ₃ alkyl, C ₁ -C ₂ fluoro-substituted alkyl, -O- (C ₁ -C ₂ ) fluoro-substituted alkyl , -O- _(C1-C3) alkyl, 8- (C ₁ -C ₃₎ alkyl, -8- _(C1-C2) fluoro-substituted alkyl, hydroxyl, amino, -ΝΗΤΟ-Ο,) alkyl and - Ν - (^ - ^) ₂ alkyl.

Monocyclic rings include 5-7 membered aryl or heteroaryl, 3-7 membered cycloalkyl and 5-7 membered non-aromatic heterocyclyl. Monocyclic rings are optionally substituted with one or more substituent groups selected from halogen, cyano, lower alkoxy, lower alkyl, hydroxyl, amino, lower alkylamino and lower dialkylamino. Examples of monocyclic groups include substituted and unsubstituted heterocycles and carbocycles. Specific monocyclic groups include thiazolyl, oxazolyl, oxazinyl, thiazinyl, dithianil, dioxanyl, isoxazolyl, isothiazolyl, triazolyl, furanyl, tetrahydrofuranyl, dihydrofuranyl, pyranyl, tetrazolyl, pyridinyl pyridyl pyridyl pyridyl pyridyl pyridyl pyridyl pyridyl piperazinyl, pyrimidinyl, morpholinyl, tetrahydrothiophenyl, thiophenyl, cyclohexyl, cyclopentyl, cyclopropyl, cyclobutyl, cycloheptanyl, azetidinyl, oxetanyl, thiranyl, oxiranyl, aziridinyl and thiomorpholinyl.

Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl and tetrazol.

Aromatic groups also include fused polycyclic aromatic ring systems in which the carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings.

Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

Fluorine substitution includes from one fluorine substituent to perfluoro substitution. Examples of fluoro-substituted C1-C2 alkyl include —CH ₂ , CP ₂ H, —CH ₃ , —CH ₂ CH ₂ P, —CH ₂ CHP ₂ , —CHRS ₃ , —CH ₂ CHP ₂ . Perfluoro-substituted C ₁ -C ₂ alkyl, for example, includes —CP ₃ and —CP ₂ CP ₃ .

Suitable substituents in the groups indicated as being substituted or unsubstituted are those which essentially do not interfere with the ability of the disclosed compound to possess one or more of the properties disclosed in this application. A substituent substantially interferes with such properties of the compound when the digital index of this property decreases by more than about 50% when combined with a substituent compared to a compound without a substituent.

Combinations of substituents and variables contemplated by the present invention are only those combinations that result in the formation of stable compounds. As used herein, the term stable refers to compounds that are stable enough to be commercially available and which maintains the integrity of the compound for a period of time that is useful for the purposes described in detail in this application.

Also included in the present invention are salts, in particular pharmaceutically acceptable salts, of sirtuin-modulating compounds described in this application. Compounds of the present invention, which contain sufficiently acidic, sufficiently basic, or both functional groups, can interact with any of a wide variety of inorganic bases and inorganic and organic acids to form a salt. Alternatively, compounds that have an inherent charge, such as quaternary nitrogen compounds, can form a salt with a suitable counterion (for example, a halide such as bromide, chloride or fluoride, in particular bromide).

The acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, secondary acid phosphate, primary acid phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suerate, sebacate, fumarate, maleate, butyn-1,4-dioate, hexin-1,6dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phosphonate, phosphonate phenylacetate, phenylpropionate, phenylbutyrate, zither t, lactate, gammagidroksibutirat, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-sulfonate, mandelate and the like.

Base addition salts include salts formed from inorganic bases such as hydroxides, carbonates, ammonium or alkali or alkaline earth metal bicarbonates and the like. Such bases useful for preparing the salts of the present invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate and the like.

According to another embodiment, the present invention provides methods for producing the sirtuin modulating compounds as defined above. Compounds can be synthesized using conventional methods. Advantageously, these compounds are conveniently synthesized from readily available starting materials.

Synthetic chemical transformations and procedures useful for synthesizing the sirtuin-modulating compounds described in this application are known in the art and include, for example, those described in K. Lateok, SotrgsNspmus Ogdashs TgaikSogtaboik (1989); T. Ogeepe apb Ρ. O.M. \ USh5. RgoYusOus Ogoyrk sh Ogdashs δγηΐΗεδίδ, 26. Eb. (1991); B. RK ^ sg apb M. RK ^ Art. Р1С5СГ apb Р1е8ег'8 Keadeik Sog Ogdats δγηίΗεδίδ (1994); and b. Radiebe, eb., Epsus 1 osoba oS Keadeik Sog Ogdats δνηΐΐιοδίδ (1995).

In an illustrative embodiment, the sirtuin-modulating compound may cross the cytoplasmic membrane of the cells. For example, a compound may have a cell penetration of at least about 20, 50, 75, 80, 90, or 95%.

The sirtuin-modulating compounds described herein may also have one or more of the following characteristics: the compound may be substantially non-toxic to the cell or subject; the sirtuin-modulating compound may be an organic molecule or a small molecule of 2,000 ashy (atomic mass units) or less, 1,000 ashy or less; the compound may have a half-life under normal atmospheric conditions of at least about 30 days, 60 days, 120 days, 6 months, or 1 year; the compound may have a half-life in solution of at least about 16,020,578 at least about 30 days, 60 days, 120 days, 6 months, or 1 year; the sirtuin-modulating compound may be more stable in solution than resveratrol at least about 50%, 2 times, 5 times, 10 times, 30 times, 50 times or 100 times; a sirtuin-modulating compound may contribute to deacetylation of the Ki70 DNA repair factor; a sirtuin-modulating compound may contribute to the deacetylation of Ke1A / p65; the compound can increase the overall metabolic rate and enhance the sensitivity of cells to ΤΝΡ-induced apoptosis.

In some embodiments, the sirtuin-modulating compound does not have any significant ability to inhibit histone deacetylase (NEAC5) class I, NEAC class II, or NEAC I and II at concentrations (e.g. w νίνο) effective to modulate the deacetylase activity of sirtuin. For example, in preferred embodiments, the sirtuin-modulating compound is a sirtuin-activating compound, and is selected so that its EC ₅₀ value for activating the deacetylase activity of sirtuin is at least 5 times less than the EC ₅₀ value for inhibiting NEAC I and / or NEAC II, and even more preferably at least 10 times, 100 times, or even 1000 times less. Methods for the analysis of SPLA I and / or SPLA II activity are well known in the art, and kits for performing such analyzes can be commercially available (see, for example, ίονίδίοη, 1ps. Sosh) and Thotak Zsheijs (Zusyshhogo. N1; уг1й У1йе уеВ а! 1ота55стот).

In some embodiments, the sirtuin-modulating compound does not possess any significant ability to modulate sirtuin homologs. In one embodiment, the activator of the human sirtuin protein may not have any significant ability to activate the lower sukuin protein of eukaryotes, in particular yeast or human pathogens, at concentrations (for example, sh uguo) effective to activate the deacetylase activity of human sirtuin. For example, a sirtuin activating compound can be selected so that its EC ₅₀ value for activating the deacetylase activity of human sirtuin, such as δΙΚΤΙ and / or δΙΚΤ3, is at least 5 times less than the EU ₅₀ value for activating yeast sirtuin, such as §ιγ2 (such as SaiFya, δ. ssgsu151ac, etc.), and even more preferably at least 10 times, 100 times, or even 1000 times less. In another embodiment, the lower eukaryotic sirtuin protein inhibitor, in particular yeast or human pathogens, does not have any significant ability to inhibit human sirtuin protein at concentrations (e.g. ίη uguo) effective to inhibit the deacetylase activity of lower eukaryotic sirtuin protein. For example, sirtuins-inhibiting compound may be chosen so that its IC ₅₀ value for inhibiting the deacetylase activity of human sirtuin, such as δΙΚΤΙ and / or δΙΚΤ3, there were at least 5 times less than the IC ₅₀ value for inhibiting sirtuin yeast such as δπ2 (such as Saiygya, δ. segeug5ge, etc.), and even more preferably at least 10 times, 100 times, or even 1000 times less.

In some embodiments, the sirtuin-modulating compound may be capable of modulating one or more homologues of the sirtuin protein, for example, one or more human δΙΚΤΙ, δΙΚΤ2, δΙΚΤ3, δΙΚΤ4, δΙΚΤ5, δΙΚΤ6, or δΙΚΤ7. In one embodiment, the sirtuin-modulating compound has the ability to modulate both δΙΚΤΙ and δΙΚΤ3 of the protein.

In other embodiments, the δΙΚΤΙ modulator does not have any significant ability to modulate other homologues of the sirtuin protein, for example, one or more human δΙΚΤ2, δΙΚΤ3, δΙΚΤ4, δΙΚΤ5, δΙΚΤ6, or δΙΚΤ7, at concentrations (for example, uguo) that are effective for modulating deacetylase human activity δΙΚΤΙ. For example, a sirtuin-modulating compound can be chosen so that its value of ΕΌ ₅₀ for modulating the deacetylase activity of human δΙΚΤΙ is at least 5 times less than the value of ΕΌ ₅₀ for modulating one or more human δΙΚΤ2, δΙΚΤ3, δΙΚΤ4, δΙΚΤ5, δΙΚΤ6 or δΙΚΤ7, and even more preferably at least 10 times, 100 times, or even 1000 times less. In one embodiment, the δΙΚΤΙ modulator does not have any significant ability to modulate the δΙΚΤ3 protein.

In other embodiments, the δΙΚΤ3 modulator does not have any significant ability to modulate other homologues of the sirtuin protein, for example, one or more human δΙΚΤΙ, δΙΚΤ2, δΙΚΤ4, δΙΚΤ5, δΙΚΤ6, or δΙΚΤ7, at concentrations (e.g. w uguo) effective to modulate deacetylase human activity δΙΚΤ3. For example, a sirtuin-modulating compound can be selected so that its value of ΕΌ ₅₀ for modulating the deacetylase activity of human δΙΚΤ3 is at least 5 times less than the value of ΕΌ ₅₀ for modulating one or more human δΙΚΤΙ, δΙΚΤ2, δΙΚΤ4, δΙΚΤ5, δΙΚΤ6 or δΙΚΤ7, and even more preferably at least 10 times, 100 times, or even 1000 times less. In one embodiment, the δΙΚΤ3 modulator does not have any significant ability to modulate the δΙΚΤ1 protein.

In some embodiments, the sirtuin modulating compound may have a binding affinity for the sirtuin protein of about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M or less. Sirtuin-17 020578 modulating compound can reduce (activator) or increase (inhibitor) the apparent CT of sirtuin protein in relation to its substrate or ΝΛΌ + (or other co-factor) in at least about 2, 3, 4, 5, 10, 20 , 30, 50 or 100 times. In some embodiments, Kt values are determined using the mass spectrometric analysis described herein.

Preferred activating compounds reduce CT of sirtuin in relation to its substrate or co-factor to a greater extent than resveratrol at the same concentration, or reduce CT of sirtuin in relation to its substrate or co-factor, similar to that achieved using resveratrol, at a lower concentration. Sirtuin-modulating compound can increase the ears of protein at least about 2, 3, 4, 5, 10, 20, 30, 50 or 100 times. The sirtuin-modulating compound may have a value of ΕΌ ₅₀ for modulating the deacetylase activity of 8ΙΚΤ1 and / or 8ΙΚΤ3 protein less than about 1 nM, less than about 10 nM, less than about 100 nM, less than about 1 μM, less than about 10 μM, less than about 100 μM, or from about 1-10 nM, from about 10100 nM, from about 0.1-1 μM, from about 1-10 μM, or from about 10-100 μM. The sirtuin-modulating compound can modulate the deacetylase activity of 8ΙΚΤ1 and / or 8ΙΚΤ3 of the protein at least about 5, 10, 20, 30, 50, or 100 times, as determined in a cell or cell based assay. Sirtuin activating compound can cause at least about 10%, 30%, 50%, 80%, 2 times, 5 times, 10 times, 50 times or 100 times greater induction of deacetylase activity of sirtuin protein compared to the same concentration resveratrol. A sirtuin-modulating compound may have a value of ΕΌ ₅₀ for modulating 8ΙΚΤ5 more at least about 10 times, 20 times, 30 times, 50 times or more than for modulating 8ΙΚΤ1 and / or 8ΙΚΤ3.

3. Application examples

In some aspects, the present invention provides methods for modulating the level and / or activity of a sirtuin protein and methods for using them.

In some embodiments, the present invention provides methods for using sirtuin modulating compounds, wherein the sirtuin modulating compounds activate the sirtuin protein, for example, increase the level and / or activity of the sirtuin protein. Sirtuin-modulating compounds that increase the level and / or activity of sirtuin protein may be useful for various therapeutic applications, including, for example, in the treatment of a person suffering from or sensitive to insulin resistance, metabolic syndrome, diabetes or its complications, or increase sensitivity to insulin in a subject. The methods include administering to a subject in need of a pharmaceutically effective amount of a sirtuin-modulating compound, for example, a sirtuin-activating compound.

Although the authors of the present invention do not wish to be bound by theory, it is believed that the activators of the present invention can interact with sirtuin at the same location in the sirtuin protein (for example, an active site or site that affects the CT or Utah of the active site). This is thought to be the reason why some classes of sirtuin activators and inhibitors may have significant structural similarities.

Sirtuin-modulating compounds described in this application can be taken separately or in combination with other compounds. A mixture of two or more sirtuin-modulating compounds can be administered to a subject in need thereof. A sirtuin-modulating compound that increases the level and / or activity of the sirtuin protein can be administered with one or more of the following compounds: resveratrol, butine, fisetin, piceatannol or quercetin. A sirtuin-modulating compound that increases the level and / or activity of sirtuin protein can be administered in combination with nicotinic acid. Also, the sirtuin-modulating compound, which reduces the level and / or activity of the sirtuin protein, can be administered with one or more of the following compounds: nicotinamide (ΝΑΜ), suramin; ΝΡ023 (antagonist Obelka); ΝΡ279 (purinergic receptor antagonist); trolox (6-hydroxy-2,5,7,8, tetramethylchroman-2carboxylic acid); (-) - epigallocatechin (hydroxy in sections 3,5,7,3 ', 4', 5 '); (-) - epigallocatechin gallate (hydroxy in sections 5,7,3 ', 4', 5 'and gallate ester in section 3); cyanidin chloride (3,5,7,3 ', 4'pentahydroxyflavilium chloride); delphinidin chloride (3,5,7,3 ', 4', 5'-hexahydroxyflavilium chloride); myricetin (cannabiscetin; 3,5,7,3 ', 4', 5'-hexahydroxyflavone); 3,7,3 ', 4', 5'-pentahydroxyflavone; gossipetin (3,5,7,8,3 ', 4'-hexahydroxyflavon), sirtinol and splitomycin. In various embodiments, combination therapies comprising a sirtuin modulating compound may refer to (1) pharmaceutical compositions that include one or more sirtuin modulating compounds in combination with one or more therapeutic agents (e.g., one or more therapeutic agents described in this application); and (2) co-administration of one or more sirtuinomodulating compounds with one or more therapeutic agents, where the sirtuinomodulating compound and therapeutic agent are not formulated in the same composition (but may be present in the same kit or package, such as a blister pack or other multi-chamber packaging; connected, individually sealed containers (for example, foil bags) that the consumer can separate; or a set where sirtuin is a modulating compound (compounds i) and other therapeutic agent (s) are in separate containers). When using separate compositions, sirtuin-modulating compound can be entered simultaneously with the introduction of another

- 18,020,578 therapeutic agents, intermittently, stepwise, before, after, or using a combination of such methods.

In some embodiments, methods for reducing the severity, prophylaxis, or treatment of diseases or disorders using a sirtuin-modulating compound may also include increasing the level of sirtuin protein, such as human 81KT1, 81KT2 and / or 81KT3 or homologues thereof. Increasing protein levels is achieved by introducing into the cell one or more copies of a nucleic acid that encodes sirtuin. For example, the level of sirtuin can be increased in a mammalian cell by introducing a nucleic acid encoding sirtuin into a mammalian cell, for example, increasing the level of 81KT1 by introducing a nucleic acid encoding the amino acid sequence represented by SeiVaik access number No. ΝΡ_036370, and / or increasing the level of 81KT3 by introducing nucleic acid encoding the amino acid sequence shown under access number SeiVaik No. AAN01042.

A nucleic acid that is introduced into the cell to increase the level of sirtuin protein can encode a protein that is at least about 80, 85, 90, 95, 98, or 99% identical to the sirtuin sequence, for example, 81KT1 and / or 81KT3 protein. For example, a nucleic acid encoding a protein can be at least about 80, 85, 90, 95, 98, or 99% identical to a nucleic acid encoding 81KT1 (for example, SeiVaik access number No. ΝΜ_12238) and / or 81KT3 (for example, SeyVaik access No. BC001042) protein. The nucleic acid may also be a nucleic acid that hybridizes, preferably under stringent hybridization conditions, with a nucleic acid encoding a wild-type sirtuin, for example, 81KT1 and / or 81KT3 protein. Stringent hybridization conditions may include hybridization and washing in 0.2 x§§C at 65 ° C. When using a nucleic acid that encodes a protein that is different from a wild-type sirtuin protein, such as a protein that is a wild-type sirtuin fragment, this protein is preferably biologically active, for example, capable of deacetylation. It is only necessary to express in the cell part of sirtuin, which is biologically active. For example, a protein that is different from wild type 81KT1 having an SeyVaik access number No. ΝΡ_036370 preferably contains a core structure thereof. The core structure sometimes refers to amino acids 62-293 with SeiVaik access number No. ΝΡ_036370, which are encoded by nucleotides 237-932 with SeiVaik access number No. nM_012238, which covers the ΝΑΌ-binding as well as substrate-binding domains. The core domain 81KT1 can also belong to the region of amino acids from 261 to 447 with SeiVaik access number No. ΝΡ_036370, which are encoded by nucleotides 834-1394 with SeiVaik access number No. nM_012238; to the amino acid region from 242 to 493 with SeiVaik access number No. ΝΡ_036370, which are encoded by nucleotides 777-1532 with SeiVaik access number No. nM_012238; or to the amino acid region from 254 to 495 with SeiVaik access number No. ΝΡ_036370, which are encoded by nucleotides 813-1538 with SeiVaik access number No. ΝΜ_012238. Whether the protein retains its biological function, for example, its ability to deacetylate, can be determined in accordance with methods known in the art.

In some embodiments, methods for reducing the severity, prophylaxis, or treatment of diseases or disorders using a sirtuin-modulating compound may also include reducing the level of sirtuin protein, such as human 81KT1, 81KT2 and / or 81KT3 or homologues thereof. A decrease in sirtuin protein level is achieved in accordance with methods known in the art. For example, κίΡΝΑ, an antisense nucleic acid or ribozyme targeted by sirtuin can be expressed in a cell. A dominant negative sirtuin mutant, for example, a mutant that is not capable of deacetylation, can also be used. For example, you can use the mutant Η363Υ protein 81KT1, described, for example, in Lio εΐ a1. (2001) Ce11 107: 137. Alternatively, agents that inhibit transcription can be used.

Methods for modulating sirtuin protein levels also include methods for modulating the transcription of genes encoding sirtuins, methods for stabilizing / destabilizing the corresponding tRNAs, and other methods known in the art.

Metabolic Disorders / Diabetes

In another aspect, sirtuin-modulating compounds that increase the level and / or activity of sirtuin protein can be used to treat or prevent a metabolic disorder such as insulin resistance, prediabetic state, type II diabetes and / or their complications. Administration of a sirtuin-modulating compound that increases the level and / or activity of the sirtuin protein may increase insulin sensitivity and / or lower insulin levels in a subject. A subject in need of such treatment may be a subject who has insulin resistance or another prior symptom of type II diabetes, who suffers from type II diabetes, or who is predisposed to develop any of these conditions. For example, a subject may be a subject that has insulin resistance, for example, high levels of insulin circulation and / or related conditions, such as hyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucose tolerance, high blood glucose, other manifestations X syndrome, hypertension, atherosclerosis, and lipodystrophy.

- 19,020,578

In an illustrative embodiment, sirtuin-modulating compounds that increase the level and / or activity of sirtuin protein can be administered as a combination therapy to treat or prevent a metabolic disorder. For example, one or more sirtuin-modulating compounds that increase the level and / or activity of sirtuin protein can be administered in combination with one or more anti-diabetic agents.

Eye disorders

One aspect of the present invention is a method of inhibiting, reducing or otherwise treating visual impairment by administering to a patient a therapeutic dose of a sirtuin modulator selected from a compound disclosed herein or a pharmaceutically acceptable salt, prodrug or metabolic derivative thereof.

In some aspects of the present invention, vision impairment is caused by retinal damage. In specific embodiments, retinal damage is caused by impaired blood flow to the eye (e.g., arteriosclerosis, vasculitis).

Examples of retinal diseases include diabetic macular edema, diabetes mellitus retinopathy, and proliferative diabetic retinopathy.

The compounds of the present invention may be useful in inhibiting treatment with a chemotherapeutic drug (e.g., a neurotoxic drug, a drug that increases intraocular pressure, such as a steroid), by administering to the subject being treated with such treatment a therapeutic dose of the sirtuin modulator disclosed in this application.

The sirtuin modulator can be administered in combination with therapy for the treatment and / or prophylaxis of SMU retinopathy.

4. Analyzes

The compounds of this application can be used for screening to identify compounds or substances that modulate sirtuins. The substance may be a nucleic acid, such as an aptamer. Assays can be performed in a cellular or non-cellular format. For example, an analysis may include incubating (or contacting) sirtuin with a test substance under conditions in which sirtuin can be modulated with an agent known as modulating sirtuin and monitoring or determining the level of sirtuin modulation in the presence of the test substance compared to the absence of the test substance. The level of sirtuin modulation can be determined by determining its ability to deacetylate the substrate. Examples of substrates include acetylated peptides, which can be obtained from VOMO (Iushoi Tyeyyid, RA). Preferred substrates include p53 peptides, such as including acetylated K382. A particularly preferred substrate is P1iotc b3-δΙΚΤΙ (VLUM), i.e. acetylated peptide Atd-Sch3-b3-b3. Other substrates are peptides from human histones NZ and H4 or acetylated amino acid. Substrates may be fluorogenic. Sirtuin may be 8ΙΚ.Τ1, δΙΚ.2, 8ΙΚ.Τ3 or part thereof. For example, recombinant 8ΙΚ.Τ1 can be obtained from VUMO. The reaction can be carried out for about 30 minutes and stopped, for example, with nicotinamide. A NEAC fluorescence activity assay / drug kit (AK-500, TYPE KEATS LABOTYUPS) can be used to determine the level of acetylation.

Similar analyzes are described in the Higher εи a1. (2002) ί. Vu1. Eat. 277: 45099. The level of sirtuin modulation in the analysis can be compared with the level of sirtuin modulation in the presence of one or more (separately or simultaneously) compounds described in this application, which can serve as positive or negative controls. Sirtuins for use in these assays may be unprocessed sirtuin proteins or parts thereof. Since it has been shown in this application that activating compounds appear to interact with the Ν-terminus of 8ΙΚ.Τ1, proteins for use in the assays include the Ν-terminal parts of sirtuins, for example, the amino acid region 1-176 or 1-255 8ΙΚ.Τ1; amino acid region 1-174 or 1-252 δίτ2.

Screening analysis includes (ΐ) contacting sirtuin with the test substance and acetylated substrate under conditions suitable for deacetylation of the substrate with sirtuin in the absence of the test substance; and (i) determining the level of substrate acetylation, where a lower level of substrate acetylation in the presence of the test substance compared to the absence of the test substance indicates that the test substance stimulates de-acetylation of sirtuin, while a higher level of substrate acetylation in the presence of the test substance compared to the absence of the test substances shows that the test substance inhibits deacetylation with sirtuin.

Methods for identifying an agent that modulates, for example, stimulates, sirtuins, can include (ΐ) contacting the cell with the test substance and substrate, which is able to penetrate the cell in the presence of a class I and class II inhibitor NEAC3 under conditions suitable for deacetylation of the substrate with sirtuin in the absence of a test substance; and (ϋ) determining the level of substrate acetylation, where a lower level of substrate acetylation in the presence of the test substance compared to the absence of the test substance indicates that the test

- 20,020,578 a substance stimulates deacetylation with sirtuin, while a higher level of substrate acetylation in the presence of a test substance compared to the absence of a test substance indicates that the test substance inhibits deacetylation with sirtuin. A preferred substrate is an acetylated peptide, which is also preferably fluorogenic, as described later in this application. The method, in addition, may include lysis of the cell to determine the level of acetylation of the substrate. Substrates can be added to the cells at a concentration in the range of from about 1 μM to about 10 mM, preferably from about 10 μM to 1 mM, even more preferably from about 100 μM to 1 mM, such as about 200 μM. A preferred substrate is acetylated lysine, for example, ε-acetyl lysine (P1ioge bb, Pbb) or P1ioge bbu88ΙΚΤ1. A preferred class I and class II inhibitor of NIAS3 is trichostatin A (ΤδΑ), which can be used at concentrations ranging from about 0.01 to 100 μM, preferably from about 0.1 to 10 μM, such as 1 μM. Incubation of the cells with the test compound and substrate can be carried out for a period of time from about 10 minutes to 5 hours, preferably about 1-3 hours. Since ΤδΑ inhibits all I class I and class II and since some substrates, for example, P1 and Be8, are poor substrate for δΓΚΤ2 and even worse substrate for δΓΚΤ3-7, such an analysis can be used to identify modulators δΓΚΤΙ w νίνο.

5. Pharmaceutical compositions

Sirtuin-modulating compounds described in this application can be formulated into the composition in the traditional way using one or more physiologically or pharmaceutically acceptable carriers or excipients. For example, sirtuin-modulating compounds and their pharmaceutically acceptable salts and solvates can be formulated for administration, for example, by injection (e.g., subcutaneous (s / c), intramuscular (i / m), intraperitoneal (and / p)), inhalation, or insufflation (either through the mouth or through the nose) or oral, buccal, sublingual, transdermal, nasal, parenteral or rectal administration. In one embodiment, the sirtuin-modulating compound can be administered topically to the site where the target cells are present, i.e. into a specific tissue, organ or body fluid (e.g., blood, cerebrospinal fluid, etc.).

Sirtuin-modulating compounds can be formulated for various modes of administration, including systemic and local or localized administration. Methods and compositions can be found in the Institute for the Development of Human Medicine. 1 δοίοποοδ. Meabe RiNZiid Co., Eurasian. RA. For parenteral administration, injections are preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For administration by injection, the compounds may be formulated in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution. In addition, the compounds can be formulated in solid form and re-dissolved or suspended immediately before use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may be in the form of, for example, tablets, lozenges, or capsules, prepared by conventional methods using pharmaceutically acceptable excipients, such as binders (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or secondary acidic calcium phosphate); lubricants (e.g. magnesium stearate, talc or silicon dioxide); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may be in the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g. lecithin or gum arabic); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl p-hydroxybenzoates or sorbic acid). The preparations may also contain buffering salts, perfumes, colorants and sweeteners, if appropriate. Preparations for oral administration can be appropriately formulated to provide a controlled release of the active compound.

For administration by inhalation (for example, for delivery to the lungs), delivery of sirtuin-modulating compounds can conveniently be accomplished using an aerosol spray from pressurized packages or a nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol preparation, the dosage unit can be determined by providing a valve for delivering a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator may be formulated as follows

- 21 020578 so that they contain a powdery mixture of the compound and a suitable base for the powder, such as lactose or starch.

Sirtuin-modulating compounds can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion. Compositions for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of a preservative. The compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

Sirtuin-modulating compounds can also be formulated in rectal compositions, such as suppositories or retained enemas, for example, containing traditional suppository bases, such as cocoa butter or other glycerides.

In addition to the compositions described above, sirtuin-modulating compounds can also be formulated as a depot of the drug. Such long-acting compositions can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, sirtuin-modulating compounds can be formulated with suitable polymeric or hydrophobic substances (for example, as an emulsion in an acceptable oil), or ion exchange resins, or as poorly soluble derivatives, for example, as a poorly soluble salt. The controlled release formula also includes patches.

Liposomes are another drug delivery system that is easily administered by injection. Accordingly, in the method of the present invention, the active compounds can also be administered in the form of a liposome delivery system. Liposomes are well known to those skilled in the art. Liposomes can be formed from various phospholipids, such as cholesterol, stearylamine phosphatidylcholines. Liposomes that can be used in the method of the present invention encompass all types of liposomes, including, but not limited to, small unilamellar vesicles, large unilamellar vesicles and multilayer vesicles.

Another way to obtain a composition, in particular a solution, a sirtuin modulator, such as resveratrol or a derivative thereof, involves the use of cyclodextrin. By cyclodextrin is meant α-, β- or γ-cyclodextrin. Cyclodextrins are described in detail in ΡιΙΗα c1 a1., US patent No. 4727064, which is incorporated into this application by reference. Cyclodextrins are cyclic glucose oligomers; these compounds form inclusion complexes with any drug whose molecule may be suitable in the lipophilic-seeking cavity of the cyclodextrin molecule.

Rapidly degradable or soluble dosage forms are useful for rapid absorption, in particular buccal and subligval absorption of pharmaceutically active agents. Fast melting dosage forms are convenient for patients, such as elderly and pediatric patients, for whom it is difficult to swallow a typical solid dosage form, such as lozenges and tablets. In addition, fast-melting dosage forms have no drawbacks associated, for example, with chewable dosage forms, where the length of time the active substance remains in the patient's mouth plays an important role in determining the amount of taste-masking substance and the degree to which the patient may reject the active substance due to an unpleasant sensation in the throat. Pharmaceutical compositions (including cosmetic preparations) may include from about 0.00001 to 100%, for example, from 0.001 to 10% or from 0.1 to 5 wt.%, One or more sirtuin-modulating compounds described in this application. In another embodiment, the pharmaceutical composition comprises: (ι) from 0.05 to 1000 mg of a compound of the present invention or a pharmaceutically acceptable salt thereof and (ϋ) from 0.1 to 2 g of one or more pharmaceutically acceptable excipients.

In one embodiment, the sirtuin modulating compound described herein is included in a topical composition containing a topical carrier which is generally suitable for topical administration of a drug and comprising any such substance known in the art. The topical carrier can be selected so as to provide the composition in the desired form, for example, in the form of an ointment, lotion, cream, microemulsion, gel, oil, solution or the like, and may consist of a substance of either natural or synthetic origin.

Preferably, the selected carrier does not adversely affect the active substance or other components of the composition for topical application. Examples of suitable topical carriers for use in this application include water, alcohols and other non-toxic organic solvents, glycerin, mineral oil, silicone, petrolatum, lanolin, fatty acids, vegetable oils, parabens, waxes and the like.

Compositions can be colorless, odorless ointments, lotions, creams, microemulsions, gels.

- 22 020578

Sirtuin-modulating compounds can be included in ointments, which, as a rule, are semi-solid preparations, usually based on petrolatum or other petroleum products. A special ointment base that can be used, as should be understood by those skilled in the art, is one that can provide optimal drug delivery and, preferably, can also provide additional desired characteristics, for example, emollient properties or the like. For other carriers or excipients, the base for the ointment should be inert, stable, non-irritating and imperceptible.

Sirtuin-modulating compounds can be incorporated into lotions, which are typically preparations for application to the skin surface without friction, and are typically liquid or semi-liquid preparations in which solid particles, including the active substance, are present in an aqueous or alcohol base. Lotions are usually suspensions of solid particles and may include an oil-in-water liquid oil emulsion.

Sirtuin-modulating compounds can be incorporated into creams, which are typically viscous liquid or semi-solid emulsions, either oil-in-water or water-in-oil. The cream base is water washable and contains an oil phase, an emulsifier and an aqueous phase. The oil phase mainly consists of petroleum jelly and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume and typically contains a humectant. The emulsifier in the composition of the cream, as explained in the above referenced document XspipCop. typically a non-toxic anionic, cationic or amphoteric surfactant.

Sirtuin-modulating compounds can be included in microemulsions, which, as a rule, are thermodynamically stable isotropically pure dispersions consisting of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Jesus Christ ^ Wagk: Magse1 Eksksg, 1992), yo1. 9).

Sirtuin-modulating compounds can be included in gel compositions, which are typically semi-solid systems consisting of either suspensions formed from small inorganic particles (two-phase systems) or large organic molecules distributed substantially uniformly in a liquid carrier (single phase gels). Although an aqueous liquid carrier is usually used for gels, alcohols and oils can also be used as a liquid carrier.

Other active substances may also be included in the composition, for example, other anti-inflammatory drugs, analgesics, antimicrobials, antifungals, antibiotics, vitamins, antioxidants and sun blocking agents commonly used in sunscreen compositions, including, but not limited to, anthranilates, benzophenones (in particular benzophenone-3), camphor derivatives, cinnamates (e.g. octylmethoxycinnamate), dibenzoylmethanes (e.g. butylmethoxybenzoylmethane), p-aminobenzoic acid (PABA) and its derivatives and salicylates (e.g., octyl salicylate).

In some topical compositions, the active substance is present in an amount that is in the range of about 0.25 to 75% by weight, based on the weight of the composition, preferably in the range of about 0.25 to 30% by weight, based on the weight composition, more preferably in the range of from about 0.5 to 15 wt.% based on the weight of the composition and most preferably in the range of from about 1.0 to 10 wt.% based on the weight of the composition.

Eye conditions can be treated or prevented, for example, systemically, topically, by intraocular injection of a sirtuin modulating compound, or by introducing a sustained release device that releases a sirtuin modulating compound. A sirtuin-modulating compound that increases the level and / or activity of the sirtuin protein can be delivered in a pharmaceutically acceptable ophthalmic carrier so as to maintain contact of the compound with the surface of the eye for a sufficient period of time, allowing the compound to penetrate into the corneal and inner regions of the eye, for example, into the anterior camera, back camera, vitreous humor, aqueous humor, endolymph, cornea, iris / ciliary region, lens, choroid / retina and sclera. A pharmaceutically acceptable ophthalmic carrier may, for example, be an ointment, a vegetable oil, or an encapsulating substance. Alternatively, the compounds of the present invention can be administered by injection directly into the endolymph or aqueous humor of the eye. In yet another alternative case, the compounds can be administered systemically, for example, by intravenous infusion or by injection, for treating the eye.

Sirtuin-modulating compounds described in this application can be stored in an environment without oxygen. For example, resveratrol or its analogue may be provided in a sealed oral capsule, such as Sar8ide1 from ΡίΐζεΓ, 1 ps.

Cells, for example, that have been treated with an ex v1yu sirtuin-modulating compound, can be administered according to methods provided for administering a transplant to a subject, which may be accompanied, for example, by the administration of an immunosuppressant, for example, cyclosporin A. General

- 23,020,578 principles for the formulation of medicines can be found in Ce11 Thégaru: 81st Ce11 Thaivriliaides, Oepe Thégars apt Ce11uhs PtipoSchgar, Le O. Mogvtoui & Lebensporis, 1996; and NstaYuroyuRs 81st Se11 Thiegar, Ε. Ό. Ba11, 1. Livsg & R. Lath, Schwibs and Hughes, 2000.

The toxicity and therapeutic efficacy of sirtuin-modulating compounds can be determined using standard pharmaceutical procedures in cell cultures or using experimental animals. LO ₅₀ is the dose lethal to 50% of the population. ΕΌ ₅₀ is the dose therapeutically effective for 50% of the population. The dose ratio causing toxic and therapeutic effects (ΕΌ ₅₀ / ΕΌ ₅₀ ) is the therapeutic index. Sirtuin-modulating compounds that exhibit large therapeutic indices are preferred. Although sirtuin-modulating compounds that exhibit toxic side effects can be used, precautionary measures must be taken and a delivery system designed to target such compounds to the affected tissue site to minimize potential damage to unaffected cells and thereby reduce side effects.

The data obtained in cell culture analyzes and animal studies can be used to formulate a series of doses intended for humans. The dose of such compounds may be in the range of circulating concentrations, which include ΕΌ ₅₀ with little or no toxicity. The dose may vary within these limits depending on the dosage form used and the route of administration used. For any compound, a therapeutically effective dose can initially be determined using cell culture assays. The dose may be formulated in animal models to achieve a plasma concentration limits which include the value of IC ₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses for humans. Plasma levels can be measured, for example, using high performance liquid chromatography.

Compounds of the present application may also be included in kits, for example, kits for therapeutic purposes. The kit may include one or more sirtuin-modulating compounds, for example, in pre-measured doses. The kit may optionally include devices for contacting the cells with the compounds and instructions for use. Devices include syringes, stents and other devices for administering a sirtuin-modulating compound to a subject (e.g., in a subject's blood vessel) or applying it to the subject's skin.

In another embodiment, the present invention provides a composition of a substance comprising the sirtuin modulator of the present invention and another therapeutic agent (those that are used in combination therapy and combination compositions) in separate dosage forms, but related to each other. The term related to each other, as used in this application, means that the individual dosage forms are packaged together or otherwise connected to each other so that it is clear that these separate dosage forms are sold and are intended for administration as part of a single treatment regimen. Such a tool and a sirtuin modulator are preferably packaged together in a blister pack or other multi-chamber packaging, or in interconnected separately sealed containers (such as foil pouches or the like) that are separated by a user (for example, tear off along an interface between two containers).

The kit may be separate vessels, including a) a sirtuin modulator of the present invention; and B) another therapeutic agent, such as the agents described herein.

For the practical implementation of the described methods use, unless otherwise indicated, the traditional procedures of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are known to specialists in this field. Such procedures are explained in detail in the literature (see, e.g., Mo1esi1ag S1otid A aogakogu Maiia1, 2 ^'1 Εά, DU Ly 8atgook, Rgvs ADF Matav (So1D 8rgshd Nagog aogakogu Rgevv: 1989); ΌΝΑ Sshtid, Uo1 I and II.... (Ό. Ν. 01Ousg ed., 1985); Odoyis1eoDe 8uyShev1v (Μ. 1. OaD ed., 1984); Mishv ek a1. I. 8. Rakeik no .: 4683195; ShsYus Assh NuPD1 / aRop (V. Ό. Natev & 8. I. ShddShv eDv. 1984); Tgaivsprkuy AiD Thaiv1aBoy (V. Ό. Natev & 8. 1. Shddvt eDv. 1984); Sbige OG AShta1 Se11v (K. I. Rzhevieu, A1ap K. L1vv, Sh., 1987); Ltbt / cd Ce11b AiD Εηζuev DKb Rgevv, 1986); B. RegLa1, and Prgasa1 OshDe That Mo1esi1ag C1otpd (1984); She Tgeabwe, MesoDw sh Nn / Utodu (AsaChild Rgevv, Sch., Ν. Υ.); Oepe TgapvGsg Ussiugv Rog Mattaap Ce11b (I. N. Msheg apD M.R. Sa1ov eDv., 1987, S1D8rgd Nagyog Lebogakogu); MECHANISM 154 APD 155 (\ Woo s! A1. Euv.) MesoDv Sh Se11 ApD Mo1esi1ag Vyuodu (Maueg anDDa1keg, eDv., AsaDetu Rgevv, LopDop, 1987); On the exhaust gas, Εxreg ^ тηηΐа1 I, by type, νо1. YU (Ό. M. \ Uss apD S.S. B1ask \ us11, eDv., 1986); Matriakshd She Moiwe Εtbguo (So1D 8rgd Nagbog Laogakogu Rgevv, So1D 8rppd Nagbog, Ν. Υ., 1986)).

- 24,020,578

Examples

The present invention, the general description of which is presented above, will be better understood by reference to the examples below, which are included solely to illustrate certain aspects and embodiments of the present invention, and are not intended to limit the present invention in any way.

Example 1. General procedure for obtaining 2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (3)

2,3-diaminobenzoic acid (1; 50 mg, 0.33 mmol), 2- (trifluoromethyl) benzaldehyde (2; 57 mg, 0.33 mmol) and sodium metabisulfite (Na ₂ 8 ₂ O ₅ , 82 mg, 0 , 43 mmol) were absorbed in ΌΜΡ (5 ml) and the mixture was stirred at 100 ° С for 17 h.

The reaction mixture was cooled to room temperature and diluted with H ₂ O. The resulting mixture was stirred at room temperature for 1 h and filtered. The collected solids were washed with water and dried to give 2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 3 as a pale brown solid (75 mg, yield: 74%). Μδ (Ε8Ι) calculated for C ' ₅ N. ₉ RA _; O ₂ : 306.06; Found: 307 [M + H].

This general procedure is used to prepare any 4-carboxy derivatives of 1H-benzo [Schimidazole using the appropriate aldehyde component as a substitute for 2 (trifluoromethyl) benzaldehyde 2 in the above scheme.

Example 2. Obtaining N- (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 208)

2- (2- (Trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (3; 75 mg, 0.24 mmol) was absorbed in ΌΜΡ (2 ml) together with thiazole-2-amine (4 ; 24 mg, 0.24 mmol) and NATI (186 mg, 0.49 mmol). ΌΙΕΑ (81 μl, 0.49 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with water and filtered. The collected solids were further purified by silica gel chromatography to give the desired product, namely Y- (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 208) as a white solid (35 mg, yield: 38%). Μδ (Ε8Ι) calculated for C ₁₈ H ₁₁ P ₃ K ₄ O8: 388.06; Found: 389 [M + H].

This general procedure is used for any amide derivatives shown in Table 1 by substitution with the appropriate carboxylic acid and amine components of compounds 3 and 4, respectively.

Example 3. Synthesis of 2- (6-piperazin-1-yl) pyridin-3-yl) -Y- (thiazol-2-yl) -1H-benzo [im] imidazole-4-carboxamide hydrochloride (compound 251).

Stage 1. Obtaining 2- (6-chloropyridin-3-yl) -1H-benzo [th] imidazole-4-carboxylic acid (6)

2,3-diaminobenzoic acid (1; 200 mg, 1.31 mmol), 6-chloronicotinaldehyde (5; 186 mg, 1.31 mmol) and Na ₂ 8 ₂ O ₅ (324 mg, 1.7 mmol) were absorbed in ΌΜΡ (10 ml) and stirred at 100 ° C for 18 h. The reaction mixture was cooled to room temperature and diluted with H ₂ O. The resulting mixture was stirred for 1 h at room temperature and filtered. The collected solids were washed with water, MeOH and dried to give 2- (6-chloropyridin-3-yl) -1Nbenzo [th] imidazole-4-carboxylic acid 6 as a pale solid (340 mg, yield: 95%).

- 25,020,578

Stage 2. Obtaining 2- (6-chloropyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (7)

2- (6-Chloropyridin-3-yl) -1H-benzo [th] imidazole-4-carboxylic acid (6; 340 mg, 1.24 mmol) was absorbed in ΌΜΡ (5 ml) together with thiazol-2-amine ( 4; 125 mg, 1.24 mmol) and NATI (943 mg, 2.48 mmol). ΌΙΕΑ (0.41 ml, 2.48 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water and the resulting solids were collected by filtration. Purification by silica gel chromatography (pentane / ETOAc) gave 2- (6-chloropyridin-3yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide 7 as a pale yellow solid (215 mg, yield: 52%). Μδ (ΕδΙ) calcd for C ₁₆ H _{1 °} S1YUO8: 355.03; Found: 356 [M + H].

Stage 3. Obtaining tert-butyl 4- (5- (4- (thiazol-2-ylcarbamoyl) -1H-benzo [y] imidazol-2-yl) pyridin-2-yl) piperazin-1-carboxylate (8)

A mixture containing 2- (6-chloropyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (7; 30 mg, 0.084 mmol) and tert-butyl piperazine- 1-carboxylate (170; 156.5 mg, 0.84 mmol) in ΌΜ8Ο (1.2 ml) was stirred at 140 ° С in a microwave reactor for 25 min. The reaction mixture was cooled to room temperature and diluted with water. The resulting solids were collected by filtration, washed with water and dried in vacuo to give tert-butyl 4- (5- (4- (thiazol-2-ylcarbamoyl) 1H-benzo [y] imidazol-2-yl) pyridin-2-yl) piperazine-1-carboxylate 8 as a pale yellow solid (20 mg, 71%). Μδ (ΕδΙ) calculated for C ₂₅ H ₂₇ SO ₃ 8: 505.19; Found: 506 [M + H].

Step 4. Preparation of 2- (6- (piperazin-1-yl) pyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [im] imidazole-4-carboxamide hydrochloride (compound 251)

A mixture containing tert-butyl 4- (5- (4- (thiazol-2-ylcarbamoyl) -1H-benzo [th] imidazol-2yl) pyridin-2-yl) piperazine-1-carboxylate (8; 20 mg, 0.039 mmol) in a methanolic solution of HCl (3N, 2 ml), stirred at room temperature for 18 hours. The precipitated solids were collected by filtration, washed with MeOH and dried to obtain 2- (6- (piperazin-1yl) pyridine- hydrochloride 3-yl) -Yu (thiazol-2-yl) -1H-benzo [y] imidazole-4-carboxamide (compound 251) as a pale yellow solid (21 mg). Μδ (ΕδΙ) calculated for С ₂₀ Н _{! 9} ЮО8 (t / ζ): 405.14; Found: 406 [M + H].

Example 4. Obtaining 2- (6-morpholinopyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 247)

A mixture containing 2- (6-chloropyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (7; 20 mg, 0.056 mmol) and morpholine (48; 49 mg, 0.56 mmol) in □ [^ O (1.5 ml), was stirred at 140 ° C in a microwave reactor for 25 minutes. The reaction mixture was cooled to room temperature and diluted with water. Precipitated solids were collected by filtration and dried. The solids were again dissolved in ° C and a sufficient amount of petroleum ether was added to precipitate the product from it again. The solids were collected by filtration, washed with MeOH and dried to give 2- (6morpholinopyridin-3-yl) -Y (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 247) as a pale a yellow solid (13 mg, 57%). Μδ (ΕδΙ) calculated for CaBdNNuGW: 406.12; Found: 407 [M + H].

- 26,020,578

Example 5. Obtaining 2- (2-morpholinopyridin-3-yl) -Y- (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 250)

2- (2-Chloropyridin-3-yl) -Y- (thiazol-2-yl) -1H-benzo [y] imidazole-4-carboxamide 9 was prepared according to the general procedure described above using the appropriate aldehyde component. A mixture containing this substance, namely 2- (2-chloropyridin-3-yl) -Y- (thiazol-2-yl) -1H-benzo [y] imidazole-4-carboxamide (9; 19 mg, 0.053 mmol) and morpholine (48; 46 mg, 0.53 mmol) in ΌΜ8Ο (1.2 ml) were stirred at 140 ° C in a microwave reactor for 25 min. The reaction mixture was cooled to room temperature and diluted with water. Precipitated solids were collected by filtration and dried. The solids were again dissolved in ° C and a sufficient amount of petroleum ether was added to precipitate the product again. The solids were collected by filtration, washed with MeOH and dried to give 2- (2-morpholinopyridin-3-yl) -Y- (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 250) as a white solid (13 mg, 60%). M8 (E8C calculated for ^ οΗι ₈ Ν ₆ Ο ₂ 8 (μ / ζ): 406.12; found: 407 [M + H].

Example 6. Obtaining 2- (2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamido) pyridine 1-oxide (compound 329)

To a solution of ^ (pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (10; 100 mg, 0.26 mmol) in CH ₂ Cl ₂ ( 10 ml) was added brCΡΒΑ (140 mg, 0.81 mmol) in nitrogen atmosphere. The mixture was stirred at room temperature for 12 hours. A saturated aqueous solution of K ₂ CO ₃ (10 ml) was added and the mixture was extracted with CH ₂ Cl ₂ (3x10 ml). The combined organic layers were dried (No. ₂ 8O ₄ ), concentrated under reduced pressure. The resulting residue was purified by chromatography (CH ₂ Cl ₂ : methanol = 20: 1) to give 2- (2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamido) pyridine 1-oxide (Compound 329) (60.0 mg, 58%) as a colorless solid. M8 (E8TS calcd for C ₂₀ H1 ^ ₃ P ₃ O _2: 398.10; found: 399 [M + H].

Example 7. Synthesis of ^ (2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazol-4-yl) thiazole-4-carboxamide (compound 332).

Stage 1. Obtaining benzyl 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazol-4-ylcarbamate (12)

Solution of 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (11; 1.53 g, 5.0 mmol), diphenylphosphoryl azide (ΌΡΡΑ, 1.39 g, 5.0 mmol) and Εΐ ₃ Ν (0.75 g, 7.4 mmol) in toluene (15 ml) were stirred at room temperature for 2 hours and then stirred at reflux for 2 hours. Benzyl alcohol (0) was added. , 54 g, 5.0 mmol) and the resulting reaction mixture was stirred at reflux for another 12 hours. After cooling to room temperature, the mixture was washed successively with aqueous citric acid to create (10 wt.% / wt.), saturated sodium ΝaΗSΟ ₃ and brine. The organic layer was dried (№ ₂ 8o ₄₎ and concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1) to obtain benzyl 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazol-4-ylcarbamate 12 (1.45 g, 70% ) as a solid. M8 (E8C calculated for C ₂₂ H ₁₆ P ^ ₃ O ₂ : 411.12; found: 412 [M + H].

- 27,020,578

Stage 2. Obtaining 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine (13)

To a stirred solution of benzyl 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4ylcarbamate (12; 1.45 g, 3.5 mmol) in methanol (20 ml) was added 5% Pb / C ( 0.20 g). The reaction mixture was thoroughly purged with nitrogen and then stirred in a hydrogen atmosphere, 1 atm, at room temperature for 18 hours. The mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to obtain 2- (3- (trifluoromethyl) phenyl) -1H-benzo [i] imidazole-4-amine 13 (0.90 g, 3.25 mmol, 93%) as a solid. Μδ (ΕδΙ) calculated for ^ ₄ Ηι ₀ Ρ ₃ Ν ₃ : 277.08; Found: 278 [M + H].

Step 3. Preparation of Y- (2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazol-4-yl) thiazole-4-carboxamide (compound 332)

A mixture of 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine (13; 0.28 g, 1.0 mmol), thiazole-4 carboxylic acid (14; 0.15 g, 1 , 2 mmol), ΌΙΕΑ (0.42 ml, 2.4 mmol) and NATI (0.79 g, 2.07 mmol) in ΌΜΡ (10 ml) were stirred at room temperature for 12 hours. A saturated aqueous NaHCO solution was added. ₃ (5 ml) and the mixture was filtered. The residue was washed with ethyl acetate (3x15 ml) and the aqueous layer was extracted with ethyl acetate (3x15 ml). The combined organic layers were dried (Μ§δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1) to obtain Y- (2- (3- (trifluoromethyl) phenyl) -1H-benzo [im] imidazole-4yl) thiazole-4-carboxamide (compound 332 ) (0.30 g, 0.77 mmol, 77%) as a colorless solid. Μδ (ΕδΙ) calculated for ^ ₈ ΗϋΡ ₃ Ν ₄ 0δ: 388.06; Found: 389 [M + H].

Example 8. Obtaining N- (pyridin-2-yl) -2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine (compound 581)

A mixture containing 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine (13; 332 mg, 1.20 mmol), 2-chloropyridine (15; 138 mg 1.22 mmol) , Pb (OAc) ₂ (120 mg, 0.53 mmol), B1YAP (336 mg, 0.53 mmol) and S-ViOK (150 mg, 1.34 mmol) in toluene (10 ml) were stirred at boiling point with reflux for 12 hours in an inert atmosphere of Ν2. The reaction mixture was concentrated under reduced pressure, and the resulting residue was extracted with ethyl acetate (3x15 ml). The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain Ν (pyridin-2-yl) -2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine ( Compound 581) (30 mg, 0.085 mmol, 7.1%) as a colorless solid. Μδ (ΕδΙ) calculated for C ₁₉ H ₁₃ P ₃ K ₄ : 354.11; found: 355 [M + H]

Example 9. Obtaining N- (pyridin-3-ylmethyl) -2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4amine (compound)

A mixture containing 2- (3- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-amine (13; 120 mg, 0.43 mmol), nicotinaldehyde (16; 51 mg, 0.47 mmol) and NaBH (OAc) ₃ (143 mg, 0.67 mmol) in 1,2-dichloroethane (10 ml) was stirred at room temperature for 12 h in an inert atmosphere of Ν ₂ . Saturated aqueous NaHCO ₃ solution (10 ml) was added and the mixture was extracted with ethyl acetate (3x15 ml). The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. Received

- 28 020578, the residue was purified by chromatography (gradient elution, petroleum ether: ethyl acetate = 1: 1 to 1: 3) to obtain ^ (pyridin-3-ylmethyl) -2- (3- (trifluoromethyl) phenyl) -1H-benzo [i] imidazole-4-amine (compound) (70 mg, 0.198 mmol, 46%) as a pale yellow solid. Μδ (ΕδΙ) calculated for Ο ₂₀ Η ₁₅ Ρ ₃ Ν ₄ : 368.12; Found: 369 [M + H].

Example 10. Synthesis of 2- (4-morpholino-2- (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [y] imidazole-4-carboxamide (compound 430).

Step 1. Preparation of 4-chloro-1- (dimethoxymethyl) -2- (trifluoromethyl) benzene (18)

To a stirred solution of 4-chloro-2- (trifluoromethyl) benzaldehyde (17; 4.20 g, 20.0 mmol) in methanol (25 ml) was added triethoxymethane (3.60 g, 24.0 mmol) at 0 ° C. The resulting reaction mixture was warmed to room temperature and stirred for 12 hours. A solution of MCO # in methanol was added to adjust pH 10. The solvent was removed under reduced pressure and the resulting residue was diluted with water. The mixture was extracted with ethyl acetate (3x50 ml) and the combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure to obtain 4-chloro-1 (dimethoxymethyl) -2- (trifluoromethyl) benzene 18 (3.20 g, 12.6 mmol, 63%).

Stage 2. Obtaining 4- (4- (dimethoxymethyl) -3- (trifluoromethyl) phenyl) morpholine (19)

A mixture of 4-chloro-1- (dimethoxymethyl) -2- (trifluoromethyl) benzene (18; 3.20 g, 12.6 mmol), Pb ₂ (ba) ₃ (0.30 g, 0.33 mmol), ΐ -ViOK (3.50 g, 31.2 mmol) and morpholine (48; 1.09 g, 12.6 mmol) in anhydrous toluene (50 ml) was stirred at reflux for 12 h in an inert atmosphere of nitrogen . After cooling to room temperature, the mixture was filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1) to give 4- (4- (dimethoxymethyl) -3- (trifluoromethyl) phenyl) morpholine 19 (0.40 g, 10.4%).

Stage 3. Obtaining 2- (4-morpholino-2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (20)

A mixture of 4- (4- (dimethoxymethyl) -3- (trifluoromethyl) phenyl) morpholine (19; 0.40 g, 1.31 mmol), 2,3-diaminobenzoic acid (1; 0.20 g, 1.31 mmol), Ν; · ι ₂ δ ₂ Ο ₅ (0.33 g, 1.74 mmol) in ΌΜΡ (10 ml) was stirred at 110 ° C (oil bath) for 18 hours. After cooling to room temperature, the mixture was diluted with water (20 ml) and stirred for 30 minutes. The mixture was filtered; the filter cake was washed with water (3x10 ml) and dried to give 2- (4-morpholino-2- (trifluoromethyl) phenyl) -1Nbenzo [th] imidazole-4-carboxylic acid 20 (0.37 g, 72%) as gray solid matter.

Step 4. Preparation of 2- (4-morpholino-2- (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 430)

Mixture of 2- (4-morpholino-2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (20; 157 mg, 0.40 mmol), ΗΑΤυ (304 mg, 0.80 mmol) , ΌΙΕΑ (207 mg, 1.60 mmol) and 3-aminopyridine (173; 37 mg, 0.40 mmol) in ΌΜΡ (2 ml) was stirred at room temperature for 12 hours.

Water (20 ml) was added and stirred for 30 minutes. The mixture was extracted with ethyl acetate (3x10 ml). The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography to give 2- (4-morpholino-2 (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [D | imidazole-4-carboxamide (compound 430) (64 mg , 34%) as a pale yellow solid. Μδ (ΕδΙ) calculated for Ο ₂₄ Η ₂₀ Ρ ₃ Ν ₅ Ο ₂ : 467.16; Found: 468 [M + H].

- 29,020,578

Example 11. Obtaining 2- (5-morpholino-2- (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 450)

The general procedure used was similar to that described in Example 10 above to obtain 2- (4 morpholino-2- (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [b] imidazole-4-carboxamide, except that 5-chloro-2- (trifluoromethyl) benzaldehyde 21 was used as starting material to give 2- (5-morpholino-2- (trifluoromethyl) phenyl) -Y- (pyridin-3-yl) -1H-benzo [b ] imidazole-4-carboxamide (compound 450). Μδ (ΕδΙ) calculated for C ₂₄ H ₂₀ P ₃ ^ O ₂ : 467.16; Found: 468 [M + H].

Example 12. Synthesis of 2- (2- (piperidin-4-yloxy) phenyl) -Y- (thiazol-2-yl) -1H-benzo [b] imidazole-4 carboxamide (compound 428).

Stage 1. Obtaining tert-butyl 4- (methylsulfonyloxy) piperidine-1-carboxylate (24)

To a solution of Y-Boc-4-hydroxypiperidine (22; 25 g, 124 mmol) and Εΐ ₃ Ν (20.7 ml) in CH ₂ Cl ₂ (100 ml) was added a solution of methanesulfonyl chloride 23 in CH ₂ Cl ₂ (20 ml) drop by drop in an ice bath. The mixture was stirred at room temperature for 18 hours. Then, the mixture was diluted with 100 ml of CH ₂ Cl ₂ and washed successively with saturated aqueous NaCO ₃ solution (50 ml) and brine (50 ml). The organic layer was dried (Al ^ O ₄ ) and concentrated under reduced pressure to obtain tert-butyl 4- (methylsulfonyloxy) piperidine-1-carboxylate 24 (45 g, crude yield:> 100%). This material was then used in the next step without further purification.

Stage 2. Obtaining tert-butyl 4- (2-formylphenoxy) piperidine-1-carboxylate (26)

A mixture containing 2-hydroxybenzaldehyde (25; 4.2 g, 34.5 mmol), tert-butyl 4- (methylsulfonyloxy) piperidine-1-carboxylate (24; 15.0 g, ~ 41 mmol), K ₂ CO ₃ (7.15 g, 51.7 mmol) and tetrabutylammonium iodide (500 mg, cat.) In MPI (100 ml) was stirred at 70-80 ° C. for 18 hours. The MPI solvent was removed by concentration under reduced pressure. The resulting residue was diluted with EUAc (100 ml) and washed with water (50 ml). The organic layer was dried (ΜдδО ₄ ) and concentrated under reduced pressure. The obtained residue was purified by chromatography (pentane / EJAc = 1: 10) to obtain tert-butyl 4- (2-formylphenoxy) piperidine-1-carboxylate 26 as a yellow oil (2.89 g, 27%).

Step 3. Preparation of 2- (2- (1- (tert-butoxycarbonyl) piperidin-4-yloxy) phenyl) -1H-benzo [b] imidazole-4-carboxylic acid (27)

The general procedure used was similar to that described above to obtain benzimidazole derivatives using 2,3-diaminobenzoic acid 1 and tert-butyl 4- (2-formylphenoxy) piperidine-1-carboxylate 26 to give 2- (2- (1- (tert -butoxycarbonyl) piperidin-4-yloxy) phenyl) 1H-benzo [b] imidazole-4-carboxylic acid 27. Μδ (ΕδΙ) calculated for C ₂₄ H ₂₇ alkali ₅ : 437.20; Found: 438 [M + H].

- 30,020,578

Step 4. Preparation of 2- (2- (piperidin-4-yloxy) phenyl) -Y- (thiazol-2-yl) -1H-benzo [D] imidazole-4 carboxamide (compound 428)

A general amide coupling procedure similar to that described above was used using 2- (2- (1 - (tert-butoxycarbonyl) piperidin-4-yloxy) phenyl) -1H-benzo [D] imidazole-4-carboxylic acid 27 and 2- aminothiazole 4 as starting materials. The resulting crude amide binding product (0.2 mmol) was treated with 4 ml of a 25% solution of ΤΡΑ in CH ₂ Cl ₂ and allowed to stand at room temperature for 8 hours. The mixture was concentrated under reduced pressure and the obtained residue was purified by preparative HPLC with using an aqueous solution of acetonitrile, buffered with 0.1% с, to obtain 2- (2- (piperidin-4-yloxy) phenyl) -Y- (thiazol-2yl) -1H-benzo [D] imidazole-4-carboxamide (compound 428). Μδ (ΕδΙ) calculated for C ₂₂ H ₂ ^ ₅ O ^: 419.14; Found: 420 [M + H].

Example 13. Synthesis of 2- (2- (pyrrolidin-3-yloxy) phenyl) -Y- (thiazol-2-yl) -1H-benzo [D] imidazole-4 carboxamide (compound 459).

Stage 1. Obtaining tert-butyl 3- (2-formylphenoxy) pyrrolidine-1-carboxylate (30)

To a solution of ^ Boc-3-hydroxypyrrolidine (28; 2 g, 10.7 mmol) and Εΐ ₃ Ν (2.2 ml, 16.0 mmol) in CH ₂ Cl ₂ (5 ml) was added a solution of methanesulfonyl chloride (1.84 g, 2.52 ml, 16.0 mmol) in CH ₂ Cl ₂ (5 ml) dropwise in an ice bath. The mixture was stirred at room temperature for 18 hours. Then the mixture was diluted with 50 ml of CH ₂ Cl ₂ , washed with saturated Na-3CO ₃ solution (20 ml), brine (20 ml), dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure to obtain Ν-Boc3- (methylsulfonyloxy) pyrrolidine 29 (2.6 g, 92%). This material was used in the next step without further purification.

2-Hydroxybenzaldehyde (25; 1.2 g, 9.8 mmol), ^ Boc-3- (methylsulfonyloxy) pyrrolidine (29; 2.6 g, 9.8 mmol), K ₂ CO ₃ (2.0 g, 14.7 mmol) and tetrabutylammonium iodide (300 mg, cat.) In CH ₂ Cl ₂ (20 ml) were stirred at 70-80 ° C for 18 hours. ΌΜΡ was removed by concentration under reduced pressure. The resulting residue was diluted with ETOAc (100 ml), washed with water (50 ml), dried (Μ§δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (pentane: EJAc = 1: 10) to obtain 676 mg of tert-butyl 3- (2-formylphenoxy) pyrrolidine-1-carboxylate 30 as an oil (yield: 23%).

Step 2. Preparation of 2- (2- (1- (tert-butoxycarbonyl) pyrrolidin-3-yloxy) phenyl) -1H-benzo [D] imidazole-4-carboxylic acid (31)

A mixture of 2,3-diaminobenzoic acid (1; 350 mg, 2.3 mmol), tert-butyl 3- (2-formylphenoxy) pyrrolidine-1-carboxylate (30; 670 mg, 2.3 mmol) and No. ^ ₂ О ₅ (568 mg, 1.3 mmol) in ΌΜΡ (30 ml) was stirred at 90 ° C for 18 hours. The mixture was cooled to room temperature. The mixture was diluted with water (100 ml), stirred for 1 h, filtered and washed with water. The filter cake was dried in vacuo to give 822 mg of the title compound 31 (84%). Μδ (ΕδΙ) calculated for C ₂₃ H ₂₅ No., O ₅ : 423.18; Found: 424 [M + H].

- 31 020578

Stage 3. Obtaining tert-butyl 3- (2- (4- (thiazol-2-ylcarbamoyl) -1H-benzo [th] imidazol-2-yl) phenoxy) pyrrolidine-1-carboxylate (32)

To a solution of 2- (2- (1- (tert-butoxycarbonyl) pyrrolidin-3-yloxy) phenyl) -1H-benzo [th] imidazole-4carboxylic acid (31; 150 mg, 0.36 mmol), thiazol-2- amine (4; 35.5 mg, 0.36 mmol) and NAUL (202 mg, 0.53 mmol) in ΌΜΡ (5 ml) was added OSA (137 mg, 1.06 mmol). The mixture was stirred at room temperature for 18 hours. The mixture was then diluted with water (20 ml) and stirred for 30 minutes. The precipitate was collected by filtration and then purified by column chromatography to obtain 85 mg of the title compound 32 (yield: 48%). Μδ (ΕδΙ) calculated for C ₂₆ H ₂₇ ^ O ^: 505.18; Found: 506 [M + H].

Step 4. Preparation of 2- (2- (pyrrolidin-3-yloxy) phenyl) ^ - (thiazol-2-yl) -1H-benzo [th] imidazole-4 carboxamide (compound 459)

A solution of tert-butyl 3- (2- (4- (thiazol-2-ylcarbamoyl) -1H-benzo [y] imidazol-2-yl) phenoxy) pyrrolidine-1-carboxylate 32 in 8 ml of 4M HCl / MeOH was stirred at room temperature for 2 hours. The resulting precipitate was collected by filtration and washed with methanol to obtain 72 mg of 2- (2 (pyrrolidin-3-yloxy) phenyl) - ^ (thiazol-2-yl) -1H-benzo [th] imidazol-4- carboxamide (compound 459) (yield: 96%). Μδ (ΕδΙ) calculated for С ^ Н ^^ ОЗЗ: 405.13; Found: 406 [M + H].

Example 14. Obtaining 2- (3- (2,3-dihydroxypropoxy) phenyl) - ^ (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 324).

Step 1. Preparation of (2,2-dimethyl-1,3-dioxolan-4-yl) methyl 4-methylbenzenesulfonate (34) but · o-4g

TaC1, Β ₃ Ν, OMAR osm.kt

ΤβοΆ'Ά

34 (2,2-Dimethyl-1, 3-dioxolan-4-yl) methanol (33; 7.56 mmol, 1.0 g) was taken up in 20 ml of dichloromethane. Then triethylamine (26.46 mmol, 3.687 ml) was added followed by ptoluenesulfonyl chloride (15.12 mmol, 2.88 g) and a catalytic amount of 4-dimethylaminopyridine. The reaction mixture was allowed to stir at room temperature for 18 hours. The crude reaction mixture was diluted with dichloromethane, washed successively with saturated aqueous sodium bicarbonate, water, dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The obtained residue 34 was used in the next step without further purification (crude yield 89%).

Stage 2. Obtaining 2- (3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) - ^ (thiazol-2-yl) -1Nbenzo [th] imidazole-4-carboxamide (36)

To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl) methyl 4-methylbenzenesulfonate (34; 4.02 mmol, 1.15 g) and 3-hydroxybenzaldehyde (35; 4.824 mmol, 0.614 g) in Potassium carbonate (10.05 mmol, 1.39 g) was added to 20 ml of dimethylformamide. The reaction mixture was stirred at 50 ° C. for 18 hours. The crude reaction mixture was diluted with water (10 ml) and extracted with dichloromethane (3x15 ml). The combined organic layers were dried (ΝΑ ₂ δΟ ₄ ) and concentrated under reduced pressure. The crude material was purified by flash chromatography (pentane / s). The desired product 36 was obtained in 84% yield.

- 32,020,578

Step 3. Preparation of 2- (3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -L- (thiazol-2-yl) -1Nbenzo [b] imidazol-4- carboxamide (37)

The general procedure similar to that described above was used to prepare benzimidazoles using 2,3-diaminobenzoic acid to give 2- (3 - ((2,2-dimethyl-1,3-dioxolan-4yl) methoxy) phenyl) -L- ( thiazol-2-yl) -1H-benzo [b] imidazole-4-carboxamide 37. Μ8 (ΕδΙ) calculated for C ₂₃ ALA ₄ O ₄ 8: 450.14; Found: 451 [M + H].

Step 4. Preparation of 2- (3- (2,3-dihydroxypropoxy) phenyl) -L- (thiazol-2-yl) -1H-benzo [b] imidazole 4-carboxamide (compound 324)

To a solution of 2- (3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -L- (thiazol-2-yl) -1H-benzo [b] imidazol-4- carboxamide (37; 50 mg, 0.111 mmol) in THF, a 4N solution of Η0 was added dropwise until a precipitate was observed. The precipitate was collected by vacuum filtration and washed with anhydrous ethanol. The product (compound 324) was dried overnight in vacuo. (The product had a pale yellow color and yield = 86%). Μδ (ΕδΙ) calculated for С ₂₃ ЩА ₄ О ₄ 8: 410,10; Found: 411 [M + H].

Example 15. Obtaining 2- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl) -L- (thiazol-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 482).

Stage 1. Obtaining 2-chloroethyl 4-methylbenzenesulfonate (39) _C1 ^^ ^he , zh _С | ^^ ^0Тз

39

In a typical experiment, p-toluenesulfonyl chloride (TgCl, 70.5 g, 0.37 mol) was added slowly to a solution of 2-chloroethanol (38; 25.0 g, 0.31 mol) in pyridine (100 ml) at 0 ° C. The reaction mixture was stirred at 0-5 ° C for 18 hours. Ice water (300 ml) was added and the resulting mixture was extracted with ethyl acetate (3x100 ml). The combined organic layers were washed with 1 N ΗΟ solution (3 × 100 ml), water (100 ml × 3), dried with Al _{2 2} О ₄ ) and concentrated under reduced pressure to obtain 2chlorethyl 4-methylbenzenesulfonate 39 (64.3 g, 88%).

Stage 2. Obtaining 2- (2-chloroethoxy) benzaldehyde (40)

A mixture of 2-chloroethyl 4-methylbenzenesulfonate (39; 27.0 g, 0.115 mol), 2-hydroxybenzaldehyde (25; 14.0 g, 0.115 mol) and K ₂ CO ₃ (31.7 g, 0.23 mol) in ΌΜΡ (100 ml) was stirred at 45 ° C for 12 hours. Water (200 ml) was added and the mixture was extracted with ethyl acetate (3x150 ml). The combined organic layers were washed with brine (3x50 mL), dried SchA ₂ 8o ₄₎ and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1) to give 2- (2-chloroethoxy) benzaldehyde 40 (16.8 g, 0.091 mol, 79%) as a pale yellow solid.

Stage 3. Obtaining 2- (2- (pyrrolidin-1-yl) ethoxy) benzaldehyde (41)

A solution of 2- (2-chloroethoxy) benzaldehyde (40; 2.5 g, 13.5 mmol), pyrrolidine (172; 1.9 g, 26.8 mmol), K ₂ CO ₃ (3.8 g, 27, 2 mmol) and ΝαΙ (0.6 g, 4.2 mmol) in ΌΜΡ (50 ml) were stirred at 80 ° C for 12 hours. Water (200 ml) was added and the mixture was extracted with ethyl acetate (3x150 ml). United organic

- 33 020578 layers were washed with brine (3x50 ml), dried (Ν; · ι ₂ δΟ. · Ι) and concentrated under reduced pressure. The obtained residue was purified by chromatography (dichloromethane: methanol = 100: 1) to obtain 2- (2- (pyrrolidin-1-yl) ethoxy) benzaldehyde 41 (1.63 g, 7.43 mmol, 55%) as a pale yellow solid.

Step 4. Preparation of 2- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl) -1H-benzo [b] imidazole-4-carboxylic acid (42)

A mixture of 2- (2- (pyrrolidin-1-yl) ethoxy) benzaldehyde (41; 1.6 g, 7.3 mmol), 2,3-diaminobenzoic acid (1; 1.10 g, 7.3 mmol), Ν ₂ δ ₂ Ο ₅ (2.0 g, 11.0 mmol) in ΌΜΡ (30 ml) was stirred at 95 ° C (oil bath) for 18 hours. Water (30 ml) was added and the mixture was filtered. The solids were washed with dichloromethane / methanol (50 ml, v / v = 5/1) and dried to give 2- (2- (2- (pyrrolidin-1yl) ethoxy) phenyl) -1H-benzo [b] imidazole 4-carboxylic acid 42 (1.80 g, 5.1 mmol, 70%) as a brown solid.

Step 5. Preparation of 2- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl) - ^ (thiazol-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 482)

Mixture of 2- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl) -1H-benzo [b] imidazole-4-carboxylic acid (42; 0.30 g, 0.85 mmol), ΗΑΤυ (0 , 42 g, 1.1 mmol), ΌΡΕΑ (0.22 g, 1.7 mmol) and 2-aminothiazole (4; 0.17 g, 1.7 mmol) in ΌΜΡ (15 ml) were stirred at 50 ° C for 14 hours. Water (30 ml) was added and the mixture was stirred for 30 minutes. The mixture was filtered and the filter cake was purified by chromatography (dichloromethane: methanol: ammonia = 60: 1: 0.3) to give 2- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl) - ^ (thiazole- 2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 482) (70 mg, 0.16 mmol, 18.8%) as a colorless solid. Μδ (Εδ ^ calculated for ^ ₃ Η ₂₃ Ν ₅ Ο ₂ δ: 433.16; found: 434 [M + H].

Example 16. Synthesis of 2- (2- (difluoromethyl) -4-fluorophenyl) -1H-benzo [b] imidazole-4-carboxylic acid (compound 559).

Stage 1. Obtaining 1-bromo-2- (difluoromethyl) -4-fluorobenzene (44)

To a solution of 2-bromo-5-fluorobenzaldehyde (43; 10 g, 43.5 mmol) in CH ₂ Cl ₂ (100 ml) was added a solution of sulfur trifluoride (diethylamino) (ΌΑδΤ, 10.5 g, 65.2 mmol) in CH ₂ Cl ₂ (50 ml) at 0 ° C. The reaction mixture was then warmed to room temperature and stirred for 18 hours. The mixture was slowly poured into an aqueous NaCl solution and the layers were separated. The aqueous layer was extracted with CH ₂ Cl ₂ . The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The crude material was purified by distillation in vacuo to give 1-bromo-2- (difluoromethyl) -4 fluorobenzene 44 (7.9 g, 71.3%).

Stage 2. Obtaining 2- (difluoromethyl) -4-fluorobenzaldehyde (46)

Isopropylmagnesium bromide (30 ml, 1M in THF, 30 mmol) was added dropwise to an ice-cooled solution of 1-bromo-2- (difluoromethyl) -4-fluorobenzene (44; 6 g, 26.7 mmol) in THF (100 ml). The reaction mixture was then allowed to warm to room temperature and stirred for 3 hours. Dimethylformamide (3.5 ml, 45.2 mmol) was added and the reaction mixture was stirred for 3 hours. Water was added and the mixture was extracted with ethyl acetate. The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography to give 2- (difluoromethyl) -4-fluorobenzaldehyde 45 (3.2 g, 68.5%).

Step 3. Preparation of 2- (2- (difluoromethyl) -4-fluorophenyl) -1H-benzo [th] imidazole-4-carboxylic acid (46)

The general procedure used was similar to that described above to obtain benzimidazole, using 2- (difluoromethyl) -4-fluorobenzaldehyde 45 and 2,3-diaminobenzoic acid 1 as starting materials to obtain 2- (2- (difluoromethyl) -4-fluorophenyl) -1H-benzo [th] imidazole-4-carboxylic acid 46. Mg (ΕδΙ) calculated for C1 ₅ H ₉ P ^ ₂ O ₂ : 306.6; Found: 307 [M + H].

Step 4. Preparation of 2- (2- (difluoromethyl) -4-fluorophenyl) ^ - (thiazol-2-yl) -1H-benzo [th] imidazole-4 carboxamide (compound 559)

A general amide coupling procedure similar to that described above was used to give 2- (2- (difluoromethyl) -4-fluorophenyl) - ^ (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 559) . Mδ (ΕδΙ) calculated for ^ ΗπΡ ^ Ν ^ δ: 388.06; Found: 389 [M + H].

Example 17. Synthesis of 2- (2- (difluoromethyl) -4-morpholinophenyl) ^ - (thiazol-2-yl) -1H-benzo [th] imidazole4-carboxamide (compound 551).

Step 1. Preparation of 2- (difluoromethyl) -4-morpholinobenzaldehyde (49)

A mixture containing 2- (difluoromethyl) -4-fluorobenzaldehyde (47; 3.50 g, 20.1 mmol), morpholine (48; 1.9 g, 22.1 mmol) and K ₂ CO ₃ (5.5 g , 40.2 mmol) in 50 ml of O1 ^ O, stirred at 100 ° C for 4 hours. The resulting reaction mixture was cooled to room temperature and diluted with water (200 ml). Precipitated solids were collected by filtration and dried to obtain 2- (difluoromethyl) -4morpholinobenzaldehyde 49 (2.5 g, 52%).

Stage 2. Preparation of 2- (2- (difluoromethyl) -4-morpholinophenyl) -1H-benzo [th] imidazole-4-carboxylic acid (50)

The general procedure used was similar to that described above for the preparation of benzimidazole, using 2- (difluoromethyl) -4-morpholinobenzaldehyde 49 and 2,3-diaminobenzoic acid 1 as starting materials to obtain 2- (2- (difluoromethyl) -4-morpholinophenyl) -1H-benzo [th] imidazole 4-carboxylic acid (50). Mδ (ΕδΙ) calculated for C ₁₉ H ₁₇ p2 ^ O ₃ : 373.12; Found: 374 [M + H].

Step 3. Preparation of 2- (2- (difluoromethyl) -4-morpholinophenyl) ^ - (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 551)

A general amide coupling procedure similar to that described above was used to give 2- (2- (difluoromethyl) -4-morpholinophenyl) - ^ (thiazol-2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 551) . Mδ (ΕδΙ) calculated for C ₂₂ H1 ₉ P ₂ ^ O ^: 455.12; Found: 456 [M + H].

Example 18. Synthesis of 2- (4-chloro-2- (trifluoromethyl) phenyl) - ^ (3- (pyrrolidin-1-ylmethyl) phenyl) -1H- 35 020578 benzo [im] imidazole-4-carboxamide (compound 490) .

Stage 1. Obtaining 3- (pyrrolidin-1-ylmethyl) aniline (53)

1- (Bromomethyl) -3-nitrobenzene (51; 5 g, 23.1 mmol) was absorbed in 100 ml of anhydrous THF together with pyrrolidine (2.3 ml, 27.72 mmol) and K ₂ CO ₃ (4.8 g 34.6 mmol). The reaction mixture was stirred at room temperature for 18 hours and then filtered. The filtrate was concentrated under reduced pressure to obtain 1- (3-nitrobenzyl) pyrrolidine 52. This substance 52 was taken up in 100 ml of absolute ΕΐΟΗ and 10% Pb / C (300 mg) was added. The resulting reaction mixture was stirred at room temperature under 1 atmosphere of hydrogen for 18 hours. The mixture was then filtered through a pad of celite and the filtrate was concentrated under reduced pressure to obtain 2.81 g of 3- (pyrrolidin1-ylmethyl) aniline 53 (70%) .

Step 2: Preparation of 2- (4-chloro-2- (trifluoromethyl) phenyl) -Y- (3- (pyrrolidin-1-ylmethyl) phenyl) -1Nbenzo [th] imidazole-4-carboxamide (compound 490)

A general amide coupling procedure similar to that described above was used to give 2- (4-chloro-2- (trifluoromethyl) phenyl) -Y- (3- (pyrrolidin-1-ylmethyl) phenyl) -1H-benzo [i] imidazole- 4 carboxamide (compound 490) from 2- (4-chloro-2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 54 and 3- (pyrrolidin-1-ylmethyl) aniline 53. Μδ (ΕδΙ) calculated for С ₂₆ Н ₂₂ С1РзЫ ₄ О: 498.14; Found: 499 [M + H].

Example 19. Synthesis of 2- (4-fluoro-2- (trifluoromethyl) phenyl) -Y- (4- (morpholinomethyl) thiazol-2-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 681).

Stage 1. Obtaining tert-butyl 4- (hydroxymethyl) thiazol-2-ylcarbamate (57)

Ethyl 2-aminothiazole-4-carboxylate (55; 10.0 g, 58.1 mmol) was absorbed in 150 ml of anhydrous THF together with di-tert-butyl carbonate (BOC ₂ O, 12.67 g, 58.1 mmol) together with 10 mg of 4- (dimethyl) aminopyridine (ΌΜΛΡ). The reaction mixture was stirred at 50 ° C. for 4 hours and then at room temperature for 18 hours. Then, the mixture was concentrated under reduced pressure to obtain a viscous oil. Pentane was added and the resulting crystalline substances were collected by filtration and dried to obtain 10.5 g of ethyl 2- (tert-butoxycarbonylamino) thiazole-4-carboxylate 56. This substance 56 (10.5 g, 38.5 mmol) was dissolved in 300 ml of anhydrous THF and dry ice-acetonitrile was cooled in a bath. Then, a solution of 1 Μ διιροΓ Nuypie ™ in THF (85 ml) was added over 10 minutes. The resulting reaction mixture was stirred at -45 ° C for 2 hours. Then, another portion of 1 Μ δηροτ Nuypye ™ in THF (35 ml) was added and the reaction mixture was stirred for another 2 hours at -45 ° C. The reaction was quenched at -45 ° C by adding 50 ml of saturated saline. After warming to room temperature, the reaction mixture was concentrated under reduced pressure. The resulting mixture was extracted with EuAc. The combined organic layers were washed with brine, dried (ΝΑ ₂ δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography to give 6.39 g of tert-butyl 4- (hydroxymethyl) thiazol-2-ylcarbamate 57 (72%).

Stage 2. Obtaining 4- (morpholinomethyl) thiazol-2-amine (59) • Voe

ΝΗ

BUT

O - o 'OH

Howl

EUN _g

- о tert-Butyl 4- (hydroxymethyl) thiazol-2-ylcarbamate (57; 2.0 g, 8.7 mmol) was absorbed in 25 ml of CH ₂ Cl ₂ together with Εί ₃ Ν (1.82 ml, 13.05 mmol) and cooled to 0 ° C. Methanesulfonyl chloride (0.85 ml,

- 36,020,578

10.88 mmol) and the resulting reaction mixture was stirred at 0 ° C for 60 minutes. Morpholine (48; 3.0 ml, 35 mmol) was then added and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. The obtained residue was taken up in ΕΐΟΑc and washed with a diluted aqueous solution of ΝаΗСΟз, saturated brine, dried (No. ₂ 8О ₄ ) and concentrated under reduced pressure. This material was purified by filtration through a short column of silica gel. The filtrate was concentrated to give 1.88 g of tert-butyl 4- (morpholinomethyl) thiazol-2-yl-carbamate 58. The second group was removed by treatment with tert-butyl 4- (morpholinomethyl) thiazol-2-yl-carbamate 58 with 20 ml 25% ΤΡΑ CH ₂ Cl ₂ for 18 hours at room temperature. After removing all solvents by concentration and drying under high vacuum, the obtained residue was treated with a pentane / ΕΐΟΑc mixture to obtain 2.17 g of 4 (morpholinomethyl) thiazole-2-amine 59 as a white solid.

Step 3. Preparation of 2- (4-fluoro-2- (trifluoromethyl) phenyl) -I- (4- (morpholinomethyl) thiazol-2-yl) -1 Nbenzo [th] imidazole-4-carboxamide (compound 681)

A general amide coupling procedure similar to that described above was used to give 2 (4-fluoro-2- (trifluoromethyl) phenyl) -H- (4- (morpholinomethyl) thiazol-2-yl) -1H-benzo [th] imcdazol-4 β-carboxamide (compound 681). M8 (E8C calculated for C ₂₃ H _{| 9} , C1P ₃ H ₂ O ₂ 8: 521.09; found: 522 [M + H].

Example 20. Synthesis of ^ (5- (morpholinomethyl) thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 780).

Stage 1. Obtaining 5- (morpholinomethyl) thiazol-2-amine (61)

5- (Morpholinomethyl) thiazole-2-amine 61 was prepared using the same synthesis sequence as described above for 4- (morpholinomethyl) thiazole-2-amine, using ethyl 2aminothiazole-5-carboxylate 60 as the starting material.

Step 2: Preparation of ^ (5- (morpholinomethyl) thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 780)

A general amide coupling procedure similar to that described above was used to give ^ (5- (morpholinomethyl) thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 780) from 2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 3 and 5 (morpholinomethyl) thiazole-2-amine 61. M8 (E8C calculated for С ₂₃ Н ₂₀ Рз ^ O ₂ 8: 487.13; found: 488 [M + H].

Example 21. Synthesis of (K) -2- (2- (difluoromethyl) -4- (2,3-dihydroxypropoxy) phenyl) -H- (thiazol-2-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 556).

Step 1. Preparation of (8) -2-bromo-5 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) benzaldehyde (64)

To a solution of 2-bromo-5-hydroxybenzaldehyde (62; 5 g, 0.025 mol) in IMR (100 ml) was added (K) -4 (chloromethyl) -2,2-dimethyl-1,3-dioxolane (63; 4, 87 g, 0.032 mol) and K ₂ CO ₃ (7, O g, 0.05 mol). The resulting reaction mixture was stirred at 150 ° C for 10 h, cooled to room temperature, diluted with water and then extracted with ΕΏΑ ^ The combined organic layers were dried (№ ₂ 8o ₄₎ and concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum ether / ΕΐΟΑc = 10: 1 to obtain the desired product, namely (8) -2-bromo-37 020578

5 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) benzaldehyde 64 (4.1 g, 56%).

Step 2. Preparation of (8) -4 - ((4-bromo-3- (difluoromethyl) phenoxy) methyl) -2,2-dimethyl-1,3-dioxolane (65)

65

To a solution of ΌΑ8Τ (3.04 g, 0.019 mol) in 20 ml of CH ₂ Cl _{2 was} added dropwise a solution of (8) -2-bromo-5 ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) benzaldehyde (64; 4.0 g, 0.013 mol) in 8 ml of CH ₂ Cl ₂ at room temperature. The resulting reaction mixture was stirred at room temperature for 10 hours. Then the mixture was diluted with CH ₂ Cl ₂ (50 ml), washed with water, dried (Na ₂ 8 O ₄ ) and concentrated under reduced pressure. Purification by chromatography (petroleum ether / ΕΐОΑс = 5: 1) gave (8) -4 - ((4-bromo-3- (difluoromethyl) phenoxy) methyl) -2,2-dimethyl-1,3-dioxolane 65 ( 3.3 g, 75%).

Step 3. Preparation of (8) -2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) benzaldehyde (66)

To a solution of (8) -4 - ((4-bromo-3- (difluoromethyl) phenoxy) methyl) -2,2-dimethyl-1,3-dioxolane (65; 3 g, 0.089 mol) in THF (30 ml) p-VcN (2.5Μ solution in hexane, 3.9 ml, 0.097 mol) was added at -78 ° C. The mixture was stirred at the same temperature for 1 h and ΌΜΡ (0.929 g, 0.103 mol) was added dropwise. After stirring at -78 ° C for another 20 minutes, a saturated aqueous solution of MH ₄ Cl (30 ml) was added. The resulting reaction mixture was warmed to room temperature and extracted with BrO (3x15 ml). The combined organic layers were dried (Haa ₂ 8O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether / ΕΐOΑc = 5: 1) to obtain the title compound, namely, (8) -2- (difluoromethyl) 4 - ((2,2-dimethyl-1,3-dioxolane -4-yl) methoxy) benzaldehyde 66 (1.83 g, 72%).

Step 4. Preparation of (8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) 1H-benzo [4] imidazol-4 -carboxylic acid (67)

To a solution of (8) -2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) benzaldehyde (66; 1.83 g, 6.40 mmol) in ΌΜΡ (150 ml) was added 2,3-diaminobenzoic acid (1; 0.97 g, 6.40 mmol) and Na ₂ 8 ₂ O ₅ (1.82 g, 9.60 mmol). The reaction mixture was stirred at 110 ° C for 18 hours. Then the mixture was cooled to room temperature and diluted with water. The resulting solids were collected by filtration and dried to give (8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4yl) methoxy) phenyl) -1H-benzo [4 ] imidazole-4-carboxylic acid 67 as a dark yellow solid (0.84 g, 51%). Μ8 (Ε8Ι) calculated for SUSHRzAOb 418.13; Found: 419 [M + H].

Step 5. Preparation of (8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) Y- (3H-pyrrole-2- il) -1H-benzo [0] imidazole-4-carboxamide (68)

(8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -1H-benzo [4] imidazole 4-carboxylic acid (67 ; 100 mg, 0.239 mmol) was absorbed in 10 ml of ΌΜΡ together with 2-aminothiazole (4; 28.7 mg, 0.289 mmol), NATI (181.7 mg, 0.478 mmol) and ΌΙΕΑ (61.8 mg, 0.289 mmol) . The resulting reaction mixture was stirred at room temperature for 12 hours and diluted with water. Precipitated solids were collected by filtration and dried. Purification by chromatography (ΕΐOΑc / hexane = 2: 1) afforded (8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl ) N- (thiazol-2-yl) -1H-benzo [0] imidazole-4-carboxamide 68 (77 mg, 65%). Μ8 (Ε8Ι) calculated for

- 38,020,578

C ₂₄ H ₂₂ P ₂ N ₄ O ₄ 8: 500.13; Found: 501 [M + H].

Step 6. Preparation of (K) -2- (2- (difluoromethyl) -4- (2,3-dihydroxypropoxy) phenyl) Y- (thiazol-2-yl) 1H-benzo [b] imidazole-4-carboxamide ( connection 556)

To a solution of (8) -2- (2- (difluoromethyl) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -Y- (thiazol2-yl) -1H- benzo [b] imidazole-4-carboxamide (68; 80 mg, 0.160 mmol) in MeOH (10 ml) was added concentrated HC1 (1 ml) and water (1 ml). The resulting reaction mixture was stirred at reflux for 2 hours. The mixture was then cooled to room temperature and a sufficient amount of 1N NaOH solution was added to adjust pH 8-9. The resulting mixture was extracted with EuAc. The combined organic layers were dried (Na ₂ 8 O ₄ ) and concentrated under reduced pressure. Purification by chromatography (petroleum ether / EJAc = 2: 1) afforded (K) -2- (2- (difluoromethyl) -4- (2,3-dihydroxypropoxy) phenyl) -Y- (thiazol-2-yl) - 1H-benzo [b] imidazole-4-carboxamide (compound 556) (67 mg, 91%). Μδ (Ε8Ι) is calculated for С ₂ 1Н ₁₈ Р ₂ Ы ₄ О ₄ 8.

Example 22. Synthesis of 6-morpholino-L- (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [b] imidazole 4-carboxamide (compound 628).

Stage 1. Obtaining 3-amino-5-chloro-2-nitrobenzoic acid (77)

To a mixture of potassium tert-butoxide (38.9 g, 0.347 mol) and copper acetate (11) monohydrate (0.9 g, 4.96 mmol) in ΌΜΡ (160 ml) was added a solution of O-methyl hydroxylamine hydrochloride (8.3 g, 99.3 mmol) and 3-chloro-6 nitrobenzoic acid (69; 10 g, 49.6 mmol) in ΌΜΡ (160 ml) at 0 ° С. The resulting reaction mixture was stirred at the indicated temperature for 3 hours. Then the mixture was quenched with water, acidified with 10% HC1 solution and extracted with EyAc. The combined organic layers were dried (No. SHO.- |) and concentrated under reduced pressure. The residue was taken up in EluAc and extracted with 10% aqueous NaOH solution. The combined aqueous layers were acidified with concentrated HCl to pH 3 and then extracted with EyAc. The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure to give 3-amino-5-chloro-2-nitrobenzoic acid 70 (6.5 g, 60.5%) as a brownish-red solid.

Stage 2. Obtaining methyl 3-amino-5-chloro-2-nitrobenzoate (71)

Concentrated H ^ O ₄ (1.5 ml) was added to a mixture of 3-amino-5-chloro-2-nitrobenzoic acid (70; 3.5 g, 16.2 mmol) in MeOH (120 ml). The resulting reaction mixture was stirred at reflux for 3 days. The mixture was then neutralized with a saturated aqueous solution of Na ₂ CO 3 and extracted with EuAc. The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. Purification by chromatography afforded methyl 3-amino-5-chloro-2-nitrobenzoate 71 (3.3 g, 88.5%) as a yellow solid.

Stage 3. Obtaining methyl 3-amino-5-morpholino-2-nitrobenzoate (72)

To a solution of 3-amino-5-chloro-2-nitrobenzoate (71; 3.3 g, 14.35 mmol) in ΌΜΡ (66 ml) was added morpholine (48; 7.12 g, 81.78 mmol) and K ₂ CO ₃ (11.28 g, 81.78 mmol). The resulting reaction mixture was stirred at 105 ° C. for 4 hours. The mixture was then filtered, diluted with water (500 ml) and extracted

- 39 020578 using ΕΏΑ ^. The combined organic layers were dried ^ a ^ OE and concentrated under reduced pressure. Column chromatography afforded methyl 3-amino-5-morpholino-2-nitrobenzoate 72 (2.1 g, 52.1%) as a brown yellow solid.

Stage 4. Preparation of 2,3-diamino-5-morpholin-4-yl-benzoic acid methyl ester (73)

A solution of methyl 3-amino-5-morpholino-2-nitrobenzoate (72; 2.1 g, 7.5 mmol) in ΕΏΗ (100 ml) was hydrogenated over 5% Pb / C (200 mg) at room temperature. After 3 hours, the reaction mixture was filtered and concentrated under reduced pressure to obtain 2,3-diamino-5-morpholin4-yl-benzoic acid methyl ester 73 (1.3 g, 69.5%) as a brown yellow solid.

Step 5. Preparation of 6-morpholin-4-yl-2- (2-trifluoromethyl-phenyl) -1H-benzoimidazole-4-carboxylic acid methyl ester (75)

To a mixture of 2,3-diamino-5-morpholin-4-yl-benzoic acid methyl ester (73; 600 mg, 2.39 mmol) in ΕΏΗ (60 ml) was added sodium metabisulfite (373 mg, 1.96 mmol), H ₂ O (2 ml) and 2 trifluoromethylbenzaldehyde (74; 990 mg, 5.69 mmol). The resulting reaction mixture was stirred at reflux for 3 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. Purification by chromatography afforded b-morpholin-4-yl-2- (2-trifluoromethyl-phenyl) -1H-benzoimidazole-4-carboxylic acid methyl ester 75 (670 mg, 69.2%) as a yellow solid.

Step 6. Preparation of 6-morpholin-4-yl-2- (2-trifluoromethyl-phenyl) -1H-benzoimidazole-4-carboxylic acid (76)

A mixture containing 6-morpholin-4-yl-2- (2-trifluoromethyl-phenyl) -1Nbenzoimidazole-4-carboxylic acid methyl ester (75; 670 mg, 1.65 mmol) in 1M NO (27 ml) was stirred with 70 ° C for 30 minutes The reaction mixture was neutralized with a 10% aqueous HC1 solution. The resulting precipitate was collected by filtration and dried to give 6-morpholin-4-yl-2- (2-trifluoromethylphenyl) -1H-benzoimidazole-4-carboxylic acid 76 (600 mg, 93%) as a pale yellow solid.

Step 7. Preparation of 6-morpholino-U (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 628)

The amide coupling procedure described in detail above was used to obtain 6 morpholino-U (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 628) from 6 -morpholin-4-yl-2- (2-trifluoromethyl-phenyl) -1H-benzoimidazole-4-carboxylic acid 76 and 2-aminothiazole 4. Μδ (ΕδΙ) calculated for C ₂₂ H1 ₈ P _s ^ O ₂ 8: 473 ,eleven; Found: 474 [M + H].

Example 23. Obtaining Yu (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -3H-imidazo [4,5-c] pyridin-7carboxamide (compound 341).

Stage 1. Preparation of 4-amino-5-nitro-nicotinic acid ethyl ester (78)

CMOE / Н ₂ 8О ₄ Ν ^ γ ^ΜΟί!

YOUNG ί ^! ^ '' ΝΗ ₂

CO ₂ E |

- 40,020,578

Potassium nitrate (20.5 g, 200 mmol) was added to an ice-cooled solution of 4-amino pyridine-3 carboxylic acid (77; 27.6 g, 200 mmol) in 200 ml of concentrated H ^ O ₄ . The resulting reaction mixture was stirred at room temperature for 20 minutes and then at 75 ° C. for 3 hours. After cooling to room temperature, ethanol (540 ml) was added with stirring. The resulting mixture was stirred at 60 ° C for 18 hours and then slowly added to an ice-cooled potassium acetate solution (800 g in 1500 ml of H ₂ O). The resulting precipitate was collected by filtration, washed with water and dried to obtain 4-amino-5-nitro-nicotinic acid ethyl ester 78 (14.6 g, 35%).

Stage 2. Preparation of 4,5-diamino-nicotinic acid ethyl ester (79)

A mixture containing 4-amino-5-nitro-nicotinic acid ethyl ester (78; 15 g, 0.071 mol) and 10% Pb / C (500 mg) in MeOH (500 ml) was stirred in a hydrogen atmosphere, 1 atm, at room temperature for 18 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated under reduced pressure to obtain 4,5-diamino-nicotinic acid ethyl ester 79 (10 g, 80%).

Step 3. Preparation of 2- (2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7carboxylic acid ethyl ester (80)

A mixture containing ethyl ester of 4,5-diamino-nicotinic acid (79; 1.81 g, 10 mmol), 2 trifluoromethylbenzaldehyde (1.9 g, 11 mmol), No. ^ ₂ O ₅ (9.5 g, 50 mmol) in 50 ml of IMR, was stirred at 120 ° C for 18 hours. After cooling to room temperature, the reaction mixture was poured into 100 ml of cold water. The resulting solids were collected by filtration, washed with water and dried to give 2- (2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid ethyl ester 80 (2.35 g, 70%) .

Step 4. Preparation of 2- (2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid (81)

2- (2-Trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid ethyl ester (80; 2.35 g, 7 mmol) was absorbed in a 10% aqueous solution of NO (40 ml ) and ethanol (20 ml). The reaction mixture was stirred at reflux for 30 minutes. After cooling to room temperature, the reaction mixture was acidified with 5N HCl. The resulting yellow solid was collected by filtration, washed with water and dried to give 2- (2 trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid 81 (1.77 g, 85%).

Step 5. Preparation of ^ (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -3H-imidazo [4,5-c] pyridin-7-carboxamide (compound 341)

The general amide coupling procedure described in detail above was used to obtain ^ (thiazol-2-yl) -2- (2- (trifluoromethyl) phenyl) -3H-imidazo [4,5-c] pyridin-7-carboxamide (compound 341 ) from 2- (2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridin-7-carboxylic acid 81 and 2-aminothiazole 4. Μδ (ΕδΙ) calculated for C17H1 ₀ PzΝ5Oδ: 389.06; Found: 390 [M + H].

Example 24. Synthesis of thiazol-2-ylamide 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid (compound 693).

- 41,020,578

Stage 1. Preparation of 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridino-carboxylic acid ethyl ester (83)

A mixture containing ethyl 4,5-diaminonicotinate (79; 0.9 g, 5 mmol) and 4-fluoro-2- (trifluoromethyl) benzaldehyde (82; 1.0 g, 5.2 mmol) in nitrobenzene (50 ml) , stirred at 220 ° C for 18 hours. After cooling to room temperature, the reaction mixture was diluted with ether. The resulting solids were collected by filtration, washed with ether and dried to give 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid ethyl ester 83 (600 mg, 39%).

Stage 2. Obtaining thiazol-2-ylamide 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid (compound 693)

A general procedure similar to that described above was used to prepare 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7-carboxylic acid thiazol-2-ylamide (compound 693) with using 2- (4-fluoro-2-trifluoromethyl-phenyl) -3H-imidazo [4,5-c] pyridine-7carboxylic acid ethyl ester 83 as a suitable starting material. Μδ (ΕδΙ) calculated for C ₁ 7Η ₉ Ρ ₄ Ν50δ: 407.05; Found: 408 [M + H].

Example 25. Synthesis of 6-methoxy-L- (pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole 4-carboxamide (compound 632).

Stage 1. Obtaining O-methylhydroxylamine (84).

Methoxylamine hydrochloride was treated with an excess of a 50% KOH solution in a small distillation apparatus for one sample. The resulting mixture was heated to 80 ° C and the fraction, which was distilled at 45-50 ° C, was collected in a receiving flask containing KOH granules.

Stage 2. Obtaining 3-amino-5-methoxy-2-nitrobenzoic acid (85)

A solution of freshly distilled o-methylhydroxylamine (3.00 g, 53.6 mmol) and 3-amino-5-methoxy-2 nitrobenzoic acid (84; 5.33 g, 27.0 mmol) in ΌΜΕ (100 ml) was added dropwise to a stirred suspension of TWion (7 equiv.) and Cu (OAc) ₂ (10 mol.%) in ΌΜΕ (50 ml) for 10 min at room temperature. After stirring for 4-8 hours at room temperature, the mixture was quenched with water and a sufficient amount of concentrated HCl was added to adjust pH 3. The resulting mixture was extracted with BYAc. The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. Purification by chromatography (CΗC1 _3- HeΟΗ-ΑcΟΗ = 8: 2: 0.1) gave 3 amino-5-methoxy-2-nitrobenzoic acid as a dark yellow solid 85 (4.62 g).

Stage 3. Obtaining 2,3-diamino-5-methoxybenzoic acid (86)

3-amino-5-methoxy-2-nitrobenzoic acid (85; 1.6 g, 7.5 mmol) was dissolved in methanol (100 ml) and 10% Pb / C (300 mg) was added. The resulting reaction mixture was stirred in an atmosphere of hydrogen, 1 atm, at room temperature for 18 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated under reduced pressure to obtain 2,3-diamino-5-methoxybenzoic acid 86 (0.7 g) .

- 42 020578

Step 4. Preparation of 6-methoxy-2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (87)

A mixture of 2,3-diamino-5-methoxybenzoic acid 86 (1.1 equiv.) And 2- (trifluoromethyl) benzaldehyde 74 (1 equiv.) And sodium metabisulfite (3 equiv.) In ΌΜΡ was stirred at reflux temperature in for 12 hours. After cooling to room temperature, the reaction mixture was diluted with water. The resulting solids were collected by filtration and dried to give 6-methoxy2- (2- (trifluoromethyl) phenyl) -1H-benzo [im] imidazole-4-carboxylic acid 87 as a dark yellow solid (40%).

Step 5. Preparation of 6-methoxy-I- (pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 632)

The amide coupling procedure described in detail above was used to prepare 6methoxy-I- (pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 632) from 6-methoxy-2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 87 and 2aminopyridine 89. Μδ (ΕδΕ) calculated for Ο ₂₁ Η ₁₅ Ρ ₃ Ν ₄ Ο ₂ : 412 ,eleven; Found: 413 [M + H].

Example 26. Synthesis of ^ (6- (pyrrolidin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1Nbenzo [th] imidazole-4-carboxamide (compound 620).

Stage 1. Obtaining ethyl 6-aminopicolinate (91)

To a solution of 2-amino-6-pyridinecarboxylic acid (90; 6.0 g, 43.5 mmol) in ethanol (150 ml) was added δΟΟ1 ₂ (12.0 g, 101 mmol) at 0 ° C. The resulting reaction mixture was stirred at reflux for 12 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. A sufficient amount of a saturated aqueous solution of Ν; · ι ₂ ίΌ _{3 was} added to adjust pH 9. The mixture was concentrated under reduced pressure, and dichloromethane (150 ml) was added to the resulting residue. The mixture was vigorously stirred at room temperature for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure to obtain ethyl 6-aminopicolinate 91 (5.5 g, 76%).

Stage 2. Obtaining ethyl 6- (tert-butoxycarbonylamino) picolinate (92)

Η ₂ Ν-ΑΙ. .CO ₂ E1

V ”

To a solution of ethyl 6-aminopycolinate (91; 5.5 g, 33 mmol) in 1-ιιΟΗ (120 ml) and acetone (40 ml) was added ΌΜΑΡ (0.08 g, 0.66 mmol) and di-tert-butyl dicarbonate (10.8 g, 49.5 mmol). The reaction mixture was stirred at room temperature for 18 hours. The solvent was removed by concentration under reduced pressure, and hexane / dichloromethane (180 ml, 3: 1) was added. The resulting mixture was cooled to −20 ° C. over 2 hours. The resulting solids were collected by filtration and dried to give ethyl 6- (tert-butoxycarbonylamino) picolinate 92 (11.0 g, 91%).

Stage 3. Obtaining tert-butyl 6- (hydroxymethyl) pyridin-2-ylcarbamate (93)

ΒοοΗΝ ^ Ν ^, ΟΟζΕί ΒοοΗΝ ^ Ν ^ ΟΗίΟΗ - V.,

To a stirred solution of ethyl 6- (tert-butoxycarbonylamino) picolinate (92; 11.0 g, 33 mmol) in THF (120 ml) in a nitrogen atmosphere was added ΡίΑ1Η ₄ (3.80 g, 100 mmol) in THF (60 ml) for 30 min at 0 ° C. The reaction mixture was stirred at 0 ° C for 6 hours and carefully quenched by adding water (2.0 ml) and 10% ΝαΟΗ solution (4.0 ml) at 0 ° C. The reaction mixture was filtered and the filtrate was dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (1: 1 petroleum ether: ethyl acetate) to give tert-butyl 6- (hydroxymethyl) pyridin-2-ylcarbamate 93 (3.0 g, 41%).

- 43,020,778

Step 4. Preparation of (6- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate (94)

VosNGk ^ CH ₂ OH MAS1 ΒοοΗΝ-, Ν ^ CH ₂ OMZ

To a solution of tert-butyl 6- (hydroxymethyl) pyridin-2-ylcarbamate (93; 3.0 g, 13.4 mmol) and ΌΙΡΕΑ (5.0 g, 40 mmol) in acetonitrile (30 ml) was added МзС1 (2, 0 g, 17.4 mmol) for 30 minutes at 0 ° C and the mixture was stirred for 2 hours at room temperature. The reaction was quenched by adding a saturated aqueous solution of ΝаΝСΟз and was extracted with ethyl acetate (3x60 ml). The combined organic layers were washed with brine, dried (# ^ O ₄ ) and concentrated under reduced pressure to give a substantially quantitative yield of crude (6- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate 94.

Stage 5. Obtaining tert-butyl 6- (pyrrolidin-1-ylmethyl) pyridin-2-ylcarbamate (96)

A mixture containing (6- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate (94; 1.30 g, 3.2 mmol), pyrrolidine (95; 0.46 g, 6.4 mmol) and K ₂ CO ₃ (1.30 g, 9.6 mmol) in acetonitrile (15 ml) was stirred at room temperature for 12 hours. A saturated aqueous solution of ΝaΗCΟ ₃ was added and the mixture was concentrated under reduced pressure. The resulting aqueous layer was extracted with ETOAc. The combined organic layers were dried (No. ^ O ₄ ) and concentrated under reduced pressure to obtain tert-butyl 6- (pyrrolidin-1-ylmethyl) pyridin-2-ylcarbamate 96 (0.75 g, 2.7 mmol, 62% for two stages).

Step 6. Preparation of 6- (pyrrolidin-1-ylmethyl) pyridin-2-amine (97)

To a solution of tert-butyl 6- (pyrrolidin-1-ylmethyl) pyridin-2-ylcarbamate (96; 750 mg, 2.7 mmol) in dichloromethane (10 ml) was added ΤΡΑ (4.0 ml) at room temperature. The resulting reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. To the resulting residue was added a sufficient amount of a saturated aqueous solution of #bCO ₃ to adjust pH 9. The mixture was then extracted with ethyl acetate (3x25 ml). The combined organic layers were dried (No. ^ O ₄ ) and concentrated under reduced pressure to obtain 6 (pyrrolidin-1-ylmethyl) pyridin-2-amine 97 (440 mg, 92%).

Step 7. Preparation of ^ (6- (pyrrolidin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1Nbenzo [D] imidazole-4-carboxamide (compound 620)

The amide coupling procedure described in detail above was used to prepare N- (6 (pyrrolidin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4 carboxamide (compound 620) from 2- (2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4-carboxylic acid 3 and 6 (pyrrolidin-1-ylmethyl) pyridin-2-amine 97. Μδ (ΕδΙ) calculated for C ₂ 5H ₂₂ P ₃ ^ O: 465.18; Found: 466 [M + H].

Example 27. Synthesis of ^ (5- (piperazin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4-carboxamide (compound 621).

Stage 1. Obtaining ethyl 6-aminonicotinate (99)

99 ^ A ^ COOH!

To a solution of 2-amino-5-pyridinecarboxylic acid (98; 5.0 g, 36.2 mmol) in ethanol (100 ml) was added δC1 ₂ (8.6 g, 72.4 mmol) at 0 ° C. The resulting reaction mixture was stirred at reflux for 12 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure, and then a sufficient amount of saturated aqueous solution No.-3CO _{3 was} added to the reaction mixture to adjust pH 9. The resulting solids collected by filtration and dried to give ethyl 6-aminonicotinate 99 (5.64 g, 94%).

- 44,020,778

Stage 2. Obtaining ethyl 6- (tert-butoxycarbonylamino) nicotinate (100) (Boc) ₂ О „ХА

LOBSTER

ΒοεΗΝ ^, Ν

SOHE!

one hundred

ΥΊι

CO _g E1

To a solution of ethyl 6-aminonicotinate (99; 11.3 g, 68.3 mmol) in ΐ-ΒиОΗ (120 ml) and acetone (40 ml) was added ΌΜΛΡ (0.17 g, 1.37 mmol) and di-tert -butyl dicarbonate (30.0 g, 137 mmol). The resulting reaction mixture was stirred at room temperature for 18 hours and then concentrated under reduced pressure. Hexane / dichloromethane (180 ml, 3: 1) was added and the reaction mixture was cooled to -20 ° C for 2 hours. The resulting solids were collected by filtration and dried to give ethyl 6- (tert-butoxycarbonylamino) nicotinate 100 (17.0) g, 68%).

Stage 3. Obtaining tert-butyl 5- (hydroxymethyl) pyridin-2-ylcarbamate (101)

1AN Rebound ~ YES

ΒοοΗΝ ^ .LC

Υ1

100 ^ '^' SOD 101 SNGON

To a stirred solution of ethyl 6- (tert-butoxycarbonylamino) nicotinate (100; 10.0 g, 27.3 mmol) in THF (40 ml) was added ILSH ₄ (1.92 g, 50.5 mmol) in THF (60 ml ) for 30 minutes at 0 ° C. The resulting reaction mixture was stirred at 0 ° C for 6 hours. The reaction was then quenched by careful addition of water (1.0 ml) and 10% NaOΗ solution (2.0 ml) at 0 ° C. The resulting mixture was filtered and the filtrate was dried ₍ 2A 8O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (dichloromethane: methanol = 40: 1) to give tert-butyl 5 (hydroxymethyl) pyridin-2-ylcarbamate 101 (4.50 g, 74%).

Step 4. Preparation of (6- (tert-butoxycarbonylamino) liridin-3-yl) methyl methanesulfonate (102)

Vosnm maa ΒοεΗΝ. .Ν

101

SNGON south

ABOUT,

CH ₂ OMa

To a mixture containing tert-butyl 5- (hydroxymethyl) pyridin-2-ylcarbamate (101; 4.5 g, 20.2 mmol) and ΌΙΡΕΑ (16.0 g, 121 mmol) in THF (45 ml) was added Μδ добав (6.93 g, 60.5 mmol) for 30 minutes at 0 ° C. The resulting reaction mixture was stirred at room temperature for 12 hours. The mixture was then washed with water (2x5 ml), dried with Al _{2 2} O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (10: 1 petroleum ether / ethyl acetate) to give (6 (tert-butoxycarbonylamino) pyridin-3-yl) methyl methanesulfonate 102 (3.0 g, 61%).

Step 5. Preparation of Benzyl 4 - ((6- (tert-butoxycarbonylamino) pyridin-3-yl) methyl) piperazine-1 carboxylate (104)

A 0 ^1M

I] lx VosNP _L ™ C:

102 kAA

A mixture of (6- (tert-butoxycarbonylamino) pyridin-3-yl) methyl methanesulfonate (102; 1.30 g, 5.4 mmol), benzylpiperazine-1-carboxylate 103 (1.42 g, 6.5 mmol), K ₂ CO ₃ (2.30 g, 16.1 mmol) and ΝαΙ (0.08 g, 0.54 mmol) in ΌΜΡ (20 ml) were stirred at 60 ° C for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. Water (30 ml) was added and the mixture was filtered. The collected solids were washed again with water and dried to give benzyl 4 - ((6- (tert-butoxycarbonylamino) pyridin-3-yl) methyl) piperazine-1-carboxylate 104 (2.20 g, 96%).

Step 6. Preparation of Benzyl 4 - ((6-aminopyridin-3-yl) methyl) piperazine-1-carboxylate (105)

To a solution of benzyl 4 - ((6- (tert-butoxycarbonylamino) pyridin-3-yl) methyl) piperazine-1 carboxylate (104; 2.20 g, 5.20 mmol) in dichloromethane (10 ml) was added TPA (3.4 g, 31.2 mmol) at room temperature.

The resulting reaction mixture was stirred at room temperature for 12 hours and then concentrated under reduced pressure. A sufficient amount of a saturated aqueous solution of # -bCO _{3 was} added to adjust pH 9. The resulting mixture was then extracted with dichloromethane (3x25 ml). The combined organic layers were dried (Μ ^^^ and concentrated under reduced pressure to give benzyl 4 - ((6-aminopyridin-3-yl) methyl) piperazine-1-carboxylate 105 (1.50 g, 89%).

- 45,020,778

Step 7. Preparation of Benzyl 4 - ((6- (2- (2- (trifluoromethyl) phenyl) -1H-benzo [b] imidazole-4-carboxamido) pyridin-3-yl) methyl) piperazine-1-carboxylate (106 )

2- (2- (Trifluoromethyl) phenyl) -1H-benzo [b] imidazole-4-carboxylic acid (3; 306 mg, 1.0 mmol) and benzyl 4 - ((6-aminopyridin-3-yl) methyl) piperazine-1-carboxylate 105 was subjected to the general amide coupling procedure described in detail above to obtain benzyl 4 - ((6- (2- (2 (trifluoromethyl) phenyl) -1H-benzo [b] imidazole-4-carboxamido) pyridine -3-yl) methyl) piperazin-1-carboxylate 106 (280 mg, 45.6%).

Step 8. Preparation of ^ (5- (piperazin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1Nbenzo [b] imidazole-4-carboxamide (compound 621)

To a solution of benzyl 4 - ((6- (2- (2- (trifluoromethyl) phenyl) -1H-benzo [b] imidazole-4-carboxamido) pyridin-3-yl) methyl) piperazine-1-carboxylate (106; 280 mg, 0.46 mmol) in methanol (20 ml) was added Rb / C (0.1 g, 5% on carbon). The reaction mixture was stirred under 1 atm H ₂ for 12 hours and then filtered through a pad of celite. The filtrate was concentrated under reduced pressure. The obtained residue was purified by chromatography (dichloromethane: methanol = 5: 1) to obtain N- (5 (piperazin-1-ylmethyl) pyridin-2-yl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [ b] imidazole-4-carboxamide (compound 621) (28.0 mg, 13%). Μδ (Εδ ^ calculated for ^ ₅ Η ₂₃ Ρ ₃ Ν ₆ Ο: 480.19; found: 481 [M + H].

Example 28. Synthesis of 2- (5-fluoro-2- (trifluoromethyl) phenyl) - ^ (5- (morpholinomethyl) pyridin-2-yl) -1Nbenzo [b] imidazole-4-carboxamide (compound 624).

Stage 1. Obtaining tert-butyl 5- (morpholinomethyl) pyridin-2-ylcarbamate (107)

A mixture containing (6- (tert-butoxycarbonylamino) pyridin-3-yl) methyl methanesulfonate (102; 1.21 g, 5.0 mmol), morpholine (48; 1.31 g, 15.0 mmol), Κ ₂ ίΌ ₃ , (2.10 g, 15.0 mmol) and NaI (0.12 g, 0.75 mmol) in ΌΜΡ (30 ml) were stirred at 80 ° C for 4 hours. After cooling to room temperature, water was added. (30 ml) and the mixture was extracted with ethyl acetate (3x30 ml). The combined organic layers were dried (Μ§δΟ ₄ ) and concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1 to 1: 1) to obtain tert-butyl 5- (morpholinomethyl) pyridin-2-ylcarbamate 107 (1.40 g, 95%).

Stage 2. Obtaining 5- (morpholinomethyl) pyridin-2-amine (108)

To a solution of tert-butyl 5- (morpholinomethyl) pyridin-2-ylcarbamate (107; 1.40 g, 4.77 mmol) in dichloromethane (10 ml) was added ΤΡΑ (10.0 ml). The mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure. A sufficient quantity of saturated aqueous solution Ν; · ι ₃ ί'Ό _3, to the resulting residue to adjust to pH 9. The mixture was extracted with dichloromethane (3x25 ml). The combined organic layers were dried (Μ§δΟ ₄ ) and concentrated under reduced pressure to give 5- (morpholinomethyl) pyridin-2-amine 108 (0.85 g, 92%).

- 46,020,578

Step 3: Preparation of 2- (5-fluoro-2- (trifluoromethyl) phenyl) - ^ (5- (morpholinomethyl) pyridin-2-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 624)

^{5OS |} g η _ζ Ν ΕΐΟΗ ²

The amide coupling procedure described in detail above was used to obtain 2- (5 fluoro-2- (trifluoromethyl) phenyl) - ^ (5- (morpholinomethyl) pyridin-2-yl) -1H-benzo [th] imidazole-4-carboxamide ( compound 624) from 5- (morpholinomethyl) pyridin-2-amine 108 and 2- (5-fluoro-2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 109. Mδ (ΕδΙ) calculated for C ₂₅ H ₂₁ P ₄ ^ O ₂ : 499.16; Found: 500 [M + H].

Example 29. Synthesis of 2- (5-fluoro-2- (trifluoromethyl) phenyl) - ^ (6- (morpholinomethyl) pyridin-3-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 686).

Stage 1. Obtaining ethyl 5-aminopicolinate (111)

110 ^ 4θ ₂ Η 111 'С0гЕ1

To a solution of 5-aminopicolinic acid (110; 8.4 g, 60.8 mmol) in ethanol (100 ml) was added δOCl ₂ (14.5 g, 120 mmol) at 0 ° C. The mixture was stirred at reflux for 12 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. A sufficient amount of a saturated aqueous solution of No.-C. O _{3 was} added to adjust pH 9. The resulting mixture was filtered and the solids were dried under reduced pressure to give ethyl 5-aminopycolinate 111 (7.5 g, 75%).

Stage 2. Obtaining ethyl 5- (tert-butoxycarbonylamino) picolinate (112) (Boc) ₂ O

Η ₂ Ν

111

СО ₂ ё (

112

CO _g E1

Vosnm ^^

112

OMAR (0.10 g, 0.9 mmol) and di-tert-butyl dicarbonate were added to a mixture of ethyl 5-aminopycolinate (111; 7.5 g, 45 mmol) in В-ViOH (60 ml) and acetone (20 ml) (19.6 g, 90 mmol). The resulting reaction mixture was stirred at room temperature for 18 hours and then concentrated under reduced pressure. Hexane (150 ml) was added and the mixture was cooled to -20 ° C for 2 hours. Precipitated solids were collected by filtration and dried to obtain ethyl 5- (tert-butoxycarbonylamino) picolinate 112 (8.9 g, 53%).

Stage 3. Obtaining tert-butyl 6- (hydroxymethyl) pyridin-3-ylcarbamate (113)

ΙΑΗ ^ΒοίΗΝ · - ^ 'ρ' СО _г Е ( ¹¹³ ^^ СНГОН

To a stirred solution of ethyl 5- (tert-butoxycarbonylamino) picolinate (112; 8.9 g, 24.0 mmol) in ethyl ether (200 ml) was added L1A1H ₄ (1.80 g, 48 mmol) in ethyl ether (100 ml ) for 30 minutes at 0 ° C. The resulting reaction mixture was stirred at 0 ° C for 3 hours and then carefully quenched by careful addition of water (1.0 ml) and 10% solution No. GON (2.0 ml). The mixture was filtered; the filtrate was dried (No. ^ O ₄ ) and concentrated under reduced pressure to obtain tert-butyl 6- (hydroxymethyl) pyridin-3-ylcarbamate 113 (4.20 g, 78%).

Step 4. Preparation of (5- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate (114) ΒοοΗΝ ^ ίχ ^ MbS1 ΒμΗΝ ^ γχ ^

113 ^ Chx% _CH20H 114

To a solution of tert-butyl 6- (hydroxymethyl) pyridin-3-ylcarbamate (113; 4.20 g, 18.8 mmol) and ΌΙΡΕΛ (7.0 g, 56.4 mmol) in THF (20 ml) was added MsCl ( 2.8 g, 24.4 mmol) for 30 minutes at 0 ° C. The resulting reaction mixture was stirred at 0 ° C for 1 h and then quenched by adding a saturated aqueous solution of ΝаΗСО ₃ . The mixture was extracted with ethyl acetate (3x60 ml). The combined organic layers were washed with brine, dried (No. ^ ₄ ) and concentrated under reduced pressure to give (5- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate 114 (5.50 g). This material was used in the next step without further purification.

- 47 020578

Stage 5. Obtaining tert-butyl 6- (morpholinomethyl) pyridin-3-ylcarbamate (115)

A mixture containing (5- (tert-butoxycarbonylamino) pyridin-2-yl) methyl methanesulfonate (114; 1.70 g), morpholine (48; 1.0 g, 11.3 mmol) and K ₂ CO ₃ (2.30 g, 16.9 mmol) in acetonitrile (30 ml), stirred at room temperature for 12 hours. Water (30 ml) was added and the mixture was extracted with ethyl acetate (3x30 ml). The combined organic layers were dried (Μ§δΟ ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ethyl acetate = 1: 1 to 1: 3) to obtain tert-butyl 6- (morpholinomethyl) pyridin-3-ylcarbamate 115 (1.20 g, 71% for two steps).

Stage 6. Obtaining 6- (morpholinomethyl) pyridin-3-amine (116)

ΒοοΗΝ

115

ShZ AkhiZ

To a solution of tert-butyl 6- (morpholinomethyl) pyridin-3-ylcarbamate (115; 1.20 g, 4.1 mmol) in dichloromethane (20 ml) was added ΤΡΑ (6.0 ml). The resulting reaction mixture was stirred at room temperature for 12 hours and then concentrated under reduced pressure. A sufficient amount of saturated aqueous solution # gC'O _{3 was} added to the residue to adjust pH 9. The mixture was extracted with ethyl acetate (3x25 ml). The combined organic layers were dried (No. ^ O ₄ ) and concentrated under reduced pressure to obtain 6- (morpholinomethyl) pyridin-3-amine 116 (450 mg, 56%).

Step 7. Preparation of 2- (5-fluoro-2- (trifluoromethyl) phenyl) - ^ (6- (morpholinomethyl) pyridin-3-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 686)

The amide coupling procedure described in detail above was used to obtain 2- (5 fluoro-2- (trifluoromethyl) phenyl) - ^ (6- (morpholinomethyl) pyridin-3-yl) -1H-benzo [th] imidazole-4-carboxamide ( compound 686) from 6- (morpholinomethyl) pyridin-3-amine 116 and 2- (5-fluoro-2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 109. Μδ (ΕδΙ) calculated for C ₂₅ H ₂₁ P ₄ ^ O ₂ : 499.16; Found: 500 [M + H].

Example 30. Synthesis of ^ (5-methylthiazol-2-yl) -2- (4- (trifluoromethyl) pyridin-3-yl) -1H-benzo [y] imidazole-4-carboxamide (compound 703).

Stage 1. Obtaining methyl 2,3-diaminobenzoate (118)

Methyl 2-amino-3-nitrobenzoate (117; 3.2 g) was dissolved in s (100 ml) and 10% Pb / C (200 mg) was added. The resulting reaction mixture was hydrogenated in an atmosphere of hydrogen, 1 ATM, at room temperature for 2 days and filtered through a celite pad. The filtrate was concentrated under reduced pressure to give a substantially quantitative yield of methyl 2,3-diaminobenzoate 118.

Stage 2. Obtaining 2- (4- (trifluoromethyl) pyridin-3-yl) -1H-benzo [th] imidazole-4-carboxylic acid (119)

4- (Trifluoromethyl) nicotinic acid (88; 1.00 g, 5.23 mmol) was absorbed in ΌΜΡ (8 ml) together with methyl 2,3-diaminobenzoate (118; 0.86 g, 5.23 mmol), NATO (2.98 g, 7.84 mmol) and OSA (1.82 ml, 10.5 mmol). The reaction mixture was stirred at room temperature for 18 hours. Then the mixture was diluted with ΕΐΟΑs, washed with water, dried (No. ^ ₄ ) and concentrated under reduced pressure to obtain crude methyl 2-amino-3- (4- (trifluoromethyl) nicotinamido ) benzoate. This material was taken up in 5 ml of glacial acetic acid and stirred at 60 ° C. for 18 hours. The reaction mixture was stirred at 90 ° C. for another 8 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. The resulting residue was diluted with EYuAs, washed with dilute aqueous ΝaNSOz, dried SchA ^ O ₄₎ and concentrated under reduced pressure to afford crude methyl 2- (4- (trifluoromethyl) pyridin-3-yl) -1Nbenzo [b] imidazol-4 -carboxylate.

This material was taken up in MeOH (8 ml) and H ₂ O (8 ml) containing PON (0.4 g) and stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure and neutralized with a 6 N HC1 solution. The resulting solids were collected by filtration and dried to give 570 mg of 2- (4- (trifluoromethyl) pyridin-3-yl) -1H-benzo [b] imidazole-4-carboxylic acid (35% yield, starting from methyl 2,3- diaminobenzoate) 119. Μδ ^ δΐ) calculated for C _{! 4} H ₈ P ₃ SC ₂ : 307.06; Found: 308 [M + H].

Step 3. Preparation of ^ (5-methylthiazol-2-yl) -2- (4- (trifluoromethyl) pyridin-3-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 703)

2- (4- (Trifluoromethyl) pyridin-3-yl) -1H-benzo [b] imidazole-4-carboxylic acid 119 was coupled with 2-amino-5-methylthiazole 171 using the general amide coupling procedure shown above, to obtain ^ (5-methylthiazol-2-yl) -2- (4- (trifluoromethyl) pyridin-3-yl) -1Nbenzo [b] imidazole-4-carboxamide (compound 703). Μδ ^ δΐ) calculated for C1 ₈ H ₁₂ P ₃ Ν50δ: 403.07; Found: 404 [M + H].

Example 31. Synthesis of 2- (2- (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) ^ - (pyridin-2-yl) -1H-benzo [b] imidazole-7-carboxamide (compound 723).

Stage 1. Obtaining 2-bromo-5- (2-morpholinoethoxy) benzaldehyde (121)

To a mixture of 2-bromo-5-hydroxybenzaldehyde (62; 3 g, 15 mmol) and 4- (2-chloroethyl) morpholinohydrochloride (120; 5.6 g, 30 mmol) in IMR (75 ml) was added K ₂ CO ₃ ( 10.3 g, 74.6 mmol). After reacting for 120 hours at 120 ° C. for 3 hours, the reaction mixture was quenched by the addition of water and extracted with EyAc. The organic layer was dried over No. ^ O ₄ and concentrated under reduced pressure. Column chromatography afforded 2-bromo-5- (2-morpholinoethoxy) benzaldehyde 121 (3.3 g, 72%) as a brown solid.

Stage 2. Obtaining 4- (2- (4-bromo-3- (difluoromethyl) phenoxy) ethyl) morpholine (123)

To a solution of 2-bromo-5- (2-morpholinoethoxy) benzaldehyde (121; 4.7 g, 15 mmol) in CH ₂ Cl ₂ (30 ml) was added a solution of ΌΑδΤ (3.63 g, 22.5 mmol) in CH ₂ C1 ₂ (15 ml) at 0 ° C. The resulting reaction mixture was stirred at reflux for 3 days. The reaction mixture was poured into a saturated aqueous solution of CaHCO ₃ and extracted with CH ₂ Cl ₂ . The combined organic layers were dried (No. ₂ 8O ₄ ) and concentrated under reduced pressure. Column chromatography afforded 4- (2- (4-bromo-3- (difluoromethyl) phenoxy) ethyl) morpholine 123 (3.13 g, 63%) as a pale yellow oil.

Stage 3. Obtaining 2- (difluoromethyl) -4- (2-morpholinoethoxy) benzaldehyde (124)

To a solution of 4- (2- (4-bromo-3- (difluoromethyl) phenoxy) ethyl) morpholine (123; 2.8 g, 8.38 mmol) in THF (90 ml) was added i-VI (1.6 M solution in hexane, 7 ml, 11.2 mmol) at -78 ° C. The resulting reaction mixture was stirred at the same temperature for 1 h and was added dropwise IMR (1.24 g,

- 49 020578 mmol). After the mixture was stirred at −78 ° C. for another 30 minutes, a saturated aqueous IN ₄ Cl solution was added and the mixture was extracted with ΕΏΑ α. The combined organic layers were dried (No. ₂ 8 O ₄ ) and concentrated under reduced pressure. Purification by chromatography afforded 2 (difluoromethyl) -4- (2-morpholinoethoxy) benzaldehyde 124 (1.6 g, 67%) as a brown yellow oil.

Step 4. Preparation of 2- (2- (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) -1H-benzo [th] imidazole-7carboxylic acid (125)

2,3-Diaminobenzoic acid (1; 222 mg, 1.5 mmol) and the corresponding 2- (difluoromethyl) -4- (2 morpholinoethoxy) benzaldehyde 124 were subjected to the same general procedure as described in detail above to obtain 2- (2- (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) -1H-benzo [th] imidazole-7carboxylic acid 125 (300 mg, 48%).

Step 5. Preparation of 2- (2- (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) -H- (pyridin-2-yl) -1Nbenzo [th] imidazole-7-carboxamide (compound 723)

The amide coupling procedure described in detail above was used to obtain 2- (2 (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) -H- (pyridin-2-yl) -1H-benzo [th] imidazole-7-carboxamide (compound 723) from 2- (2- (difluoromethyl) -4- (2-morpholinoethoxy) phenyl) -1H-benzo [th] imidazole-7carboxylic acid 125 and 2-aminopyridine 89. M8 (E8C calculated for C ₂₅ H ₂₁ P ₄ H ₅ O ₂ : 499.16; Found: 500 [M + H].

Example 32. Synthesis of (K) -2- (4- (2,3-dihydroxypropoxy) -2- (trifluoromethyl) phenyl) -H- (pyridin-2-yl) 1H-benzo [th] imidazole-4-carboxamide ( compound 757).

Step 1. Preparation of (8) -4 - ((4-bromo-3- (trifluoromethyl) phenoxy) methyl) -2,2-dimethyl-1,3-dioxolane (127)

(8) -4- (Chloromethyl) -2,2-dimethyl-1,3-dioxolane (63; 3.3 g, 20.5 mmol) was added to the mixture containing 4-bromo-3- (trifluoromethyl) phenol ( 126; 5 g, 20 mmol) and K ₂ CO ₃ (8.30 g, 60 mmol) in 20 ml of IMR. The resulting reaction mixture was stirred at 60 ° C for 6 hours. After cooling to room temperature, the mixture was poured into water and extracted with ΕΏΑ α. The combined organic layers were dried (No. ₂ 8 O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ΕΐΟΑc = 50: 1) to give (8) -4 - ((4-bromo-3 (trifluoromethyl) phenoxy) methyl) -2,2-dimethyl-1,3-dioxolane (4 g, 56%).

Step 2. Preparation of (8) -4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) benzaldehyde (128)

A solution of PVAH in hexane (6.76 ml, 1.6 M in hexane) was added dropwise to a solution of (8) -4 - ((4-bromo-3 (trifluoromethyl) phenoxy) methyl) -2,2-dimethyl-1 , 3-dioxolane (127; 3.20 g, 9 mmol) in THF (50 ml) at 78 ° C for 30 min. After stirring for an additional 10 minutes, MIP (1.4 ml) was added dropwise over 10 minutes. The resulting reaction mixture was warmed to room temperature and stirred for 30 minutes. The mixture was then quenched with water and extracted with ΕΏΑα. The combined organic layers were dried (No. ₂ 8O ₄ ) and concentrated under reduced pressure. The obtained residue was purified by chromatography (petroleum ether: ΕΐΟΑc = 50: 1) to obtain (8) -4 - ((2,2dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) benzaldehyde 128 .

Step 3. Preparation of (8) -2- (4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) phenyl) 1H-benzo [th] imidazole-4 -carboxylic acid (129)

- 50,020,578

^) - 4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) benzaldehyde 128 was reacted with 2,3-diaminobenzoic acid 1, following a general procedure similar to that described above, to obtain ^) - 2- (4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) phenyl) -1Nbenzo [th] imidazole-4-carbon acids 129.

Step 4. Preparation of (K) -2- (4- (2,3-dihydroxypropoxy) -2- (trifluoromethyl) phenyl) -H- (pyridin-2yl) -1H-benzo [th] imidazole-4-carboxamide (compound 757)

^) - 2- (4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole 4-carboxylic acid 129 was reacted with 2-aminopyridine 89 using the same reaction sequence as described in example 25, step 5, to obtain (K) -2- (4- (2,3-dihydroxypropoxy) -2- (trifluoromethyl) phenyl) -Y- (pyridine -2-yl) -1H-benzo [th] imidazole-4-carboxamide (compound 757). Μδ (ΕδΙ) calculated for C ₂₃ H19p ₃ S ₅ O ₄ : 472.14; Found: 473 [M + H].

Example 33. Synthesis of ^) - M- (3- (2,3-dihydroxypropoxy) phenyl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 725).

Step 1. Preparation of (K) -2,2-dimethyl-4 - ((3-nitrophenoxy) methyl) -1,3-dioxolane (132)

3-Nitrophenol (130; 2.0 g, 14.37 mmol) was absorbed in 20 ml of anhydrous ΌΜΡ together with anhydrous potassium carbonate (4.96 g, 35.93 mmol) and (K) -4- (chloromethyl) -2 , 2-dimethyl-1,3-dioxolane (131; 2.55 ml, 18.68 mmol). The resulting reaction mixture was heated in a microwave reactor with stirring at 160 ° C. for 4 hours. The crude reaction mixture was washed with water, filtered and extracted with dichloromethane (3x15 ml). The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography using ethyl acetate: pentane to give the desired product 132 as an amber-colored oil (52%).

Step 2. Preparation of (K) -3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) aniline (133)

In a nitrogen atmosphere, Pe powder (2.38 g, 42.54 mmol) and YH ₄ Cl (2.38 g, 42.54 mmol) were combined, followed by the addition of (K) -2,2-dimethyl-4 - (( 3-nitrophenoxy) methyl) -1,3-dioxolane (132; 1.8 g, 7.09 mmol) and a 4: 1 mixture of isopropanol: water (30 ml: 10 ml). The reaction mixture was stirred at reflux for 18 hours. The crude material was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The resulting aqueous layer was extracted with dichloromethane (3x15 ml). The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure to obtain (K) -3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) aniline 133 (1.2 g , 79% yield). This material was used in the next step without any further purification.

- 51 020578

Step 3. Preparation of (K) -№ (3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [ d] imidazole-4-carboxamide (134)

2- (2- (Trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid (3; 75 mg, 0.24 mmol) was absorbed in ΝΜΡ (2 ml) together with (K) -3- ( (2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) aniline (133; 64.3 mg, 0.288 mmol) and NATi (186 mg, 0.49 mmol). ΌΙΕΑ (81 μl, 0.49 mmol) was added and the resulting reaction mixture was stirred at room temperature for 12 hours. The temperature was then raised to 60 ° C and the reaction mixture was allowed to stir for another 1 hour. The reaction mixture was diluted with water and filtered. The collected solids were further purified by silica gel chromatography to give the desired product 134, as a yellow solid (51.5 mg, 42%).

Step 4. Preparation of ^) - ^ (3- (2,3-dihydroxypropoxy) phenyl) -2- (2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 725)

To a solution of (K) - ^ (3 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -2- (2- (trifluoromethyl) phenyl) 1H-benzo [th] imidazole -4-carboxamide (134; 51.5 mg, 0.1 mmol) in 3 ml of THF was added a 4 N HC1 solution dropwise until it was possible to observe a color change, the solution was dark, at this point another 1 ml of HC1 was added and the solution was allowed to stir at room temperature for 18 hours. The reaction mixture was diluted with MeOH and then concentrated under reduced pressure. The resulting yellowish suspension was purified by preparative HPLC. The desired product (Compound 725) was obtained as a white solid (32.5 mg, 64%). Μδ (ΕδΙ) calculated for C ₂₄ H ₂₀ P ^ ₃ O ₄ : 471.14; Found: 472 [M + H].

Example 34. Synthesis of 2- (3- (2,6-dimethylmorpholino) phenyl) ^ - (thiazol-2-yl) -1H-benzo [th] imidazole-4 carboxamide (compound 576).

Stage 1. Obtaining 2- (3-bromophenyl) -1,3-dioxane (136)

135 136

A mixture containing 3-bromobenzaldehyde (135; 18.5 g, 100 mmol), propane-1,3-diol (9.1 g, 120 mmol) and TkOH (0.1 g) in toluene (200 ml) was stirred at the boiling temperature under reflux in a Dean-Stark trap for 18 hours. After cooling to room temperature, the mixture was washed with a 5% aqueous solution of No.-C. O ₃ (200 ml), dried (No. ^ O ₄ ) and concentrated under reduced temperature. pressure to obtain a substantially quantitative yield of crude 2- (3-bromophenyl) -1,3 dioxane 136 as a yellow oil (25 g).

Stage 2. Obtaining 3- (2,6-dimethylmorpholino) benzaldehyde (137)

A mixture containing 2- (3-bromophenyl) -1,3-dioxane (136; 2.0 g, 8.23 mmol), 2,6-dimethylmorpholine (122; 1.14 g, 9.88 mmol), Ry ₂ (dLa) ₃ (50 mg), No. L1-Vi (1.39 g, 14.0 mmol) and ΒιΝΑΡ (100 mg) in 20 ml of toluene were stirred at reflux for 5 hours. After cooling A sufficient amount of a cold solution of 1N HCl (50 ml) was added to room temperature to adjust pH 1. The resulting reaction mixture was stirred at room temperature for 1 h. No. JUN (aq) was added to adjust pH 11. The aqueous mixture was extracted with CH ₂ C1 ₂ (3x50 ml). The combined organic layers were dried (M ^ O ₄ ) and concentrated under reduced pressure. Purification by chromatography (petroleum ether: ΕΐОΑс = 10: 1) gave 3- (2,6dimethylmorpholino) benzaldehyde 137 as a yellow oil (1.36 g, yield: 72%).

Step 3. Preparation of 2- (3 - ((2,6-dimethylmorpholino) phenyl) -1H-benzo [4] imidazole-4-carboxylic acid (138)

2,3-Diaminobenzoic acid (1; 0.94 g, 6.2 mmol) and 3- (2,6-dimethylmorpholino) benzaldehyde (137; 1.36 g, 6.2 mmol) were subjected to the same general procedure as described above, to obtain 2- (3 ((2,6-dimethylmorpholino) phenyl) -1H-benzo [4] imidazole-4-carboxylic acid 138 in 87% yield (1.9 g).

Step 4. Preparation of 2- (3- (2,6-dimethylmorpholino) phenyl) -I- (thiazol-2-yl) -1H-benzo [4] imidazole-4 carboxamide (compound 576)

The amide coupling procedure described in detail above was used to prepare 2- (3 (2,6-dimethylmorpholino) phenyl) -I- (thiazol-2-yl) -1H-benzo [0] imidazole-4-carboxamide (compound 576) from 2- (3 - ((2,6-dimethylmorpholino) phenyl) -1H-benzo [4] imidazole-4-carboxylic acid 138 and 2-aminothiazole 4. Μ8 (Ε8Ι) calculated for C ₂₃ H ₂₅ P ₂ H ₅ O ₃ : 433.16; found: 434 [M + H].

Example 35. Synthesis of (K) -2- (2- (difluoromethyl) phenyl) -I- (3- (2,3-dihydroxypropoxy) phenyl) -6-fluoro1H-benzo [4] imidazole-4-carboxamide (compound 811 )

Stage 1. Obtaining 5-fluoro-2- (2,2,2-trifluoroacetamido) benzoic acid (140)

2-amino-5-fluorobenzoic acid (139; 1.0 g, 6.4 mmol) was taken up in 10 ml of anhydrous THF and the resulting mixture was cooled to 0 ° C. Then 2,2,2-trifluoroacetic anhydride (3.6 ml) was added dropwise at 0 ° C. The resulting reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure. The residue was treated with ice cooling and the resulting precipitate was collected by filtration, washed with water and dried to give 5-fluoro-2- (2,2,2 trifluoroacetamido) benzoic acid 140 (1.3 g, 80%).

Stage 2. Obtaining 5-fluoro-3-nitro-2- (2,2,2-trifluoroacetamido) benzoic acid (141)

5-Fluoro-2- (2,2,2-trifluoroacetamido) benzoic acid (140; 1.29 g, 5.1 mmol) was added in small portions to fuming nitric acid (7 ml) at 0 ° C. The resulting reaction mixture was stirred at 0 ° C for 1 h and then poured into ice water (10 ml). The resulting precipitate was collected by filtration, washed with water and dried to obtain 141 (1.2 g, 80%) 5-fluoro-3-nitro-2- (2,2,2 trifluoroacetamido) benzoic acid.

Stage 3. Obtaining 2-amino-5-fluoro-3-nitrobenzoic acid (142)

To a solution of 5-fluoro-3-nitro-2- (2,2,2-trifluoroacetamido) benzoic acid (141; 1.2 g, 4.1 mmol) in ethanol (20 ml) was added a 10% aqueous solution of sodium hydroxide ( 20 ml). The resulting reaction mixture was stirred at 80 ° C. for 3 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The resulting orange solid was acidified — 53,020,578 — using diluted HCl, filtered and dried to give 2-amino-5-fluoro-3-nitrobenzoic acid 142 (0.73 g, 90%).

Stage 4. Obtaining 2,3-diamino-5-fluorobenzoic acid (143)

2-amino-5-fluoro-3-nitrobenzoic acid (142; 0.73 g) was dissolved in methanol (10 ml). To this solution was added 5% Pb / C (0.1 g). The resulting reaction mixture was stirred at room temperature in an atmosphere of hydrogen, 1 atm, for 18 hours and then filtered through a celite pad. The filtrate was concentrated under reduced pressure to obtain 2,3-diamino-5-fluorobenzoic acid 143 as a brown solid (0.59 g, 95%).

Step 5. Preparation of 2- (2- (difluoromethyl) phenyl) -5-fluoro-1H-benzo [th] imidazole-7-carboxylic acid (144)

2,3-Diamino-5-fluorobenzoic acid (143; 0.826 g, 5.29 mmol) and 2- (difluoromethyl) benzaldehyde 144 were subjected to the same general procedure as described above to obtain 2- (2- (difluoromethyl) phenyl ) 5-fluoro-1H-benzo [th] imidazole-7-carboxylic acid 145, which was obtained in 65% yield (0.7 g). Μδ (Εδΐ) calculated for С ^ НдР ^ Оу 306.6; Found: 307 [M + H].

Step 6. Preparation of ^^ - ^ - (difluoromethyl ^ enylCJ ^ - ^ D-dimethyl-C3-dioxolan-CH-yl) methoxy) phenyl) -6-fluoro-1H-benzo [th] imidazole-4-carboxamide (147)

2- (2- (Difluoromethyl) phenyl) -5-fluoro-1H-benzo [th] imidazole-7-carboxylic acid (145; 250 mg, 0.82 mmol) was combined with ^ -3- ^ D-dimethyl- C3-dioxolanCH-yl-ethoxyCniline 146 (prepared as described for 133 in Example 33, Step 1-2, except for using (8) -4- (chloromethyl) -2,2 dimethyl-1,3-dioxolane in Step 1), as described above. (§) -2- (2- (Difluoromethyl) phenyl) -№ (3 - ((2,2dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -6-fluoro-1H-benzo [th] imidazole-4-carboxamide 147 was obtained in 48% yield (200 mg). Μδ (ΕδΙ) calculated for C ₂₇ H ₂₄ P ^ ₃ O ₄ : 511.17; Found: 512 [M + H].

Step 7. Preparation of (K) -2- (2- (difluoromethyl) phenyl) -Y (3- (2,3-dihydroxypropoxy) phenyl) -6-fluoro1H-benzo [th] imidazole-4-carboxamide (compound 811)

Ten drops of concentrated HCl were added to a solution of ^^ - ^ - (difluoromethyl ^ enylCUR ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) phenyl) -6-fluoro-1H-benzo [th] imidazole -4-carboxamide (147; 200 mg, 0.391 mmol) in methanol (15 ml). The resulting reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure to obtain (K) -2- (2 (difluoromethyl) phenyl) -Y (3- (2,3-dihydroxypropoxy) phenyl) -6-fluoro -1H-benzo [th] imidazole-4-carboxamide (compound 811) (160 mg, 87%). Μδ (ΕδΙ) calculated for C ₂₇ H ₂₄ P ₃ ^ O ₄ : 511.17; Found: 512 [M + H].

Example 36. Synthesis of 2- (2-methylpyridin-3-yl) -Y (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [y] imidazole-4-carboxamide (compound 814).

- 54,020,778

Stage 1. Obtaining 2-methylnicotinaldehyde (149)

To a solution of 3-bromo-2-methylpyridine (148; 10 g, 58.1 mmol) in THF (150 ml) was added p-VI (2.5 M, 25.6 ml) at -78 ° C. The reaction mixture was stirred at this temperature for 1 hour. Then, ΌΜΡ (1.30 ml) was added and the resulting reaction mixture was stirred for 1 hour at -78 ° C. The reaction was quenched by the addition of an aqueous solution of OH ₄ Cl. After warming to room temperature, the mixture was extracted with EuAc. The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography to give 2-methylnicotinaldehyde 149 (2.18 g, 31%).

Stage 2. Obtaining 2- (2-methylpyridin-3-yl) -1H-benzo [b] imidazole-4-carboxylic acid (150)

2-Methylnicotinaldehyde (149; 2.18 g, 18 mmol) was subjected to standard conditions used to prepare substituted benzimidazoles by reaction with 2,3-diaminobenzoic acid 1. Desired product, 2- (2-methylpyridin-3-yl) - 1H-benzo [b] imidazole-4-carboxylic acid 150, was obtained by purification using chromatography, followed by crystallization from ESAc (0.43 g, 11%).

Step 3. Preparation of 2- (2-methylpyridin-3-yl) -Y- (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 814)

2- (2-Methylpyridin-3-yl) -1H-benzo [b] imidazole-4-carboxylic acid 150 was combined with

6-morpholinomethylpyridin-2-amine 172 (prepared as described in Example 29, Steps 1-6, except using ethyl 6-aminopycolinate in Step 1) using the standard amide coupling procedure described in detail above to give 2- (2 -methylpyridin-3-yl) -Y- (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 814). Μδ (ΚδΙ) calculated for C ₂₄ H ₂₄ N ₆ O ₂ : 428.20; Found: 429 [M + H].

Example 37. Synthesis of 2- (6-methylpyridin-3-yl) -Y- (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 815).

Stage 1. Obtaining 6-methylnicotinaldehyde (152)

161 152

To a solution of 5-bromo-2-methylpyridine (151; 10 g, 58.1 mmol) in THF (150 ml) was added i-CuE1 (2.5 M, 25.6 ml) at -78 ° C. The reaction mixture was stirred at this temperature for 1 hour. Then, ΌΜΡ (1.30 ml) was added and the resulting reaction mixture was stirred for 1 hour at -78 ° C. The reaction was quenched by the addition of an aqueous solution of OH ₄ Cl. After warming to room temperature, the mixture was extracted with EuAc. The combined organic layers were dried (Na ^ O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography to give 15-methylnicotinaldehyde 152 (5.0 g, 72%).

Step 2. Preparation of 2- (6-methylpyridin-3-yl) -Y- (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 815)

6-Methylnicotinaldehyde 152 was combined with 2,3-diaminobenzoic acid 1 using the same standard conditions described in detail above to give the corresponding substituted benzimidazole 153. The resulting 2- (6-methylpyridin-3-yl) - 1H-benzo [b] imidazole-4-carboxylic acid 153 was combined with 6-morpholinomethylpyridin-2-amine 172 using the same standard amide coupling procedure to give 2- (6-methylpyridin-3-yl) ^ (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 815). Μδ (Εδ ^ calculated for ^ ₄ Η ₂₄ Ν ₆ Ο ₂ : 428.20; found: 429 [M + H].

Example 38. Synthesis of 2- (2-methylpyridin-4-yl) ^ - (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 816).

Stage 1. Obtaining 2-methylisonicotinaldehyde (156)

To a solution of 2,4-lutidine (154; 10 g, 93.3 mmol) in THF (150 ml) was added p-VI (2.5 M, 41.1 ml) at -78 ° C. Then diethylamine (8.19 g, 112 mmol) was added at the same temperature, followed by ΌΜΡ (10 ml). The resulting reaction mixture was stirred at -78 ° C for 1 h. The reaction was quenched by adding an aqueous solution of ΝΗ ₄ Ο. After warming to room temperature, the mixture was extracted with HMC1 ₂ . The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure to give intermediate enamine 155.

To a solution SHYU) (40 g) in water (200 mL) was added the above enamine intermediate SSCHS1 155 in ₂ (200 mL). The reaction mixture was stirred at room temperature for 18 hours. Then was added a sufficient amount of 2N ΝαΟΗ solution to adjust the pH of the mixture to 8. The mixture was then filtered, separated and extracted with ₂ SSCHS1. The combined organic layers were dried (No. ₂ δΟ ₄ ) and concentrated under reduced pressure. Purification by chromatography afforded 2 methylisonicotinaldehyde 156 (4 g, 35%).

Step 2: Preparation of 2- (2-methylpyridin-4-yl) ^ - (6- (morpholinomethyl) pyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 816)

2-Methylisonicotinaldehyde 156 was combined with 2,3-diaminobenzoic acid 1 using the same standard conditions as described above, to give 2- (2methylpyridin-4-yl) -1H-benzo [b] imidazole-4-carboxylic acid 157, which was then combined with 6-morpholinomethylpyridin-2-amine 172 using the same standard amide coupling procedure to give 2- (2-methylpyridin-4-yl) ^ - (6- (morpholinomethyl) pyridin-2-yl) -1 Nbenzo [b] imidazole-4-carboxamide (compound 816). Μδ (Εδ ^ calculated for ^ ₄ Η ₂₄ Ν ₆ Ο ₂ : 428.20; found: 429 [M + H].

Example 39. Synthesis of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) - ^ (thiazol-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 828).

Step 1. Preparation of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [b] imidazole 4-carboxylic acid (159)

2- (4-Chloro-2- (trifluoromethyl) phenyl) -1H-benzo [b] imidazole-4-carboxylic acid (54; 500 mg, 1.46 mmol) was suspended in ССС1 ₂ (15 ml). Methanol (2 ml) was added to dissolve. Oxalyl chloride (130 μl, 1.46 mmol) was added dropwise to the reaction solution. After stirring the reaction mixture at room temperature overnight, the mixture was concentrated. The residue was absorbed in ° C and washed successively with saturated Na растворомCΗz solution and then with saturated saline. The organic layer was dried over Μ§δΟ ₄ , filtered and concentrated. Purification by chromatography on silica gel ^ ϊΟΑ ^ ε ^ ά ^ gave methyl 2- (4-chloro-2- (trifluoromethyl) phenyl) -1Nbenzo [b] imidazole-4-carboxylate as an orange solid (structure not shown) ( 390 mg, yield: 75%).

From a sealed tube containing a mixture of methyl 2- (4-chloro-2- (trifluoromethyl) phenyl) 1H-benzo [b] imidazole-4-carboxylate (390 mg, 1.10 mmol), potassium 1-trifluoroborate-ethylpyrrolidine

- 56 020578 (158; 212 mg, 1.11 mmol), Pb (OLs) ₂ (22 mg, 0.03 mmol), XRp (31 mg, 0.06 mmol) and cesium carbonate (1.0 g, 3 , 3 mmol) pumped gas and filled with nitrogen twice. THF / H ₂ O (10: 1, 4.4 ml) was added to the mixture. The suspension was heated at 80 ° C. overnight and cooled to room temperature. Water (2 ml) was added and the reaction mixture was extracted with ΠΗ ₂ Ο ₂ twice. The combined organic layers were washed with brine, dried over MgSO ₄ , filtered and concentrated. Purification by chromatography on silica gel (MeO ^^^^^ Εΐ ₃ Ν) gave methyl 2- (4 (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [i] imidazole-4-carboxylate (structure not shown) as a yellow solid (544 mg).

Methyl 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [ά] imidazole-4-carboxylate (544 mg) and ^^ ΟΗ (95 mg, 4 mmol) were dissolved in THF / H ₂ O (1: 1, 24 ml). The reaction mixture was stirred overnight at room temperature, then heated at 50 ° C. overnight. Solvents were removed in vacuo. The residue was taken up in water and the pH was adjusted to 7-8 with a 1N solution of ΗΟ. The mixture was allowed to stir for 1 h, then the solids were collected by filtration to obtain 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 159 in the form of a pale yellow solid (170 mg, yield in 2 stages: 40%). M8 (Ε8Ι) calculated for Ο ^^ Ν ^: 389.14; Found: 390 [M + H].

Step 2: Preparation of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y- (thiazol-2-yl) -1Nbenzo [th] imidazole-4-carboxamide (compound 828)

To a solution of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4 carboxylic acid (159; 60 mg, 0.154 mmol), 2-aminothiazole (4; 17 mg, 0.169 mmol) and ΗΑΤυ (117 mg, 0.308 mmol) in ΌΜΡ (1.5 ml) was added diisopropylethylamine (107 μl, 0.616 mmol). The reaction mixture was microwaved at 140 ° C for 15 minutes. The reaction mixture was diluted with EAAc and washed sequentially with water and brine. The organic layer was dried over MgSO ₄ , filtered and concentrated. Purification by preparative HPLC and subsequent lyophilization with a dilute aqueous solution of ала gave the 2- (4 (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y- (thiazol-2-yl) -1H-benzo ΗΟ-salt [i] imidazole-4-carboxamide (compound 828) as a brownish solid (24.3 mg, yield: 31%). M8 (Ε8Ι) calculated for C ₂₃ Η ₂₀ Ρ ₃ Ν5Ο8: 471,13; Found: 472 [M + H].

Example 40. Synthesis of Y- (pyridin-2-yl) -2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1Nbenzo [th] imidazole-4-carboxamide (compound 838)

2- (4- (Pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 159 was combined with 2-aminopyridine 89 using the general procedure similar to that described above in Example 39 to afford Y- (pyridin-2-yl) -2- (4- (pyrrolidin-1-ylmethyl) -2 (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 838) . M8 (Ε8Ι) calculated for C ^^ P ^ O: 465.18; Found: 466 [M + H].

Example 41. Synthesis of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y- (4- (pyrrolidin-1-ylmethyl) phenyl) -1H-benzo [th] imidazol-4- carboxamide (compound 869)

2- (4- (Pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [th] imidazole-4-carboxylic acid 159 was combined with 4- (pyrrolidin-1-ylmethyl) aniline 160 to obtain 2- (4 (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y- (4- (pyrrolidin-1-ylmethyl) phenyl) -1H-benzo [th] imidazole-4-carboxamide (compound 869) using the general procedure similar to that described in example 39. M8 (Ε8Ι) was calculated for C ^^ P ^ O: 547.26; Found: 548 [M + H].

Example 42. Synthesis of 2- (4- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y-p-tolyl-1H-benzo [y]

- 57 020578 imidazole-4-carboxamide (compound 870)

2- (4- (Pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4-carboxylic acid 159 was combined with p-toluidine 161 to obtain 2- (4- (pyrrolidin- 1-ylmethyl) -2 (trifluoromethyl) phenyl) -Y-p-tolyl-1H-benzo [D] imidazole-4-carboxamide (compound 870) using the general procedure similar to that described in example 39. M8 (Ε8 ^ calculated for C ₂₇ H ₂₅ P ₃ ^ O: 478.20; Found: 479 [M + H].

Example 43. Synthesis of 2- (5- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) ^ - (thiazol-2-yl) -1Nbenzo [D] imidazole-4-carboxamide (compound 871)

The general procedure used was similar to that described in Example 39 above, steps 1 and 2, except that 2- (5-chloro-2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4 carboxylic acid 163 was used as starting material to give 2- (5- (pyrrolidin-1-ylmethyl) -2 (trifluoromethyl) phenyl) -Y- (thiazol-2-yl) -1H-benzo [D] imidazole-4-carboxamide (compound 871) . M8 (Ε8 ^ calculated for C ₂₃ H ₂₀ P ₃ ^ O8: 471.13; found: 472 [M + H].

Example 44. Synthesis of ^ (pyridin-2-yl) -2- (5- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1Nbenzo [D] imidazole-4-carboxamide (compound 872)

The general procedure used was similar to that described above in Example 39, stage 1, and Example 40, except that 2- (5-chloro-2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4 carboxylic acid was used 163 as starting material, to obtain ^ (pyridin-2-yl) -2- (5 (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4-carboxamide (compound 872 ) M8 (Ε8 ^ calculated for C ₂₅ H ₂₂ P ₃ ^ O: 465.18; found: 466 [M + H].

Example 45. Synthesis of 2- (5- (pyrrolidin-1-ylmethyl) -2- (trifluoromethyl) phenyl) -Y- (4- (pyrrolidin-1-ylmethyl) phenyl) -1H-benzo [D] imidazol-4- carboxamide (compound 873)

The general procedure used was similar to that described above in Example 39, stage 1, and Example 41, except that 2- (5-chloro-2- (trifluoromethyl) phenyl) -1H-benzo [D] imidazole-4-carboxylic acid was used 163 as starting material, to give 2- (5- (pyrrolidin-1-ylmethyl) -2 (trifluoromethyl) phenyl) -Y- (4- (pyrrolidin-1-ylmethyl) phenyl) -1H-benzo [D] imidazole- 4-carboxamide (compound 873). M8 (Ε8 ^ calculated for C ₃ 1H ₃₂ P ₃ ^ O: 547.26; found: 548 [M + H].

Example 46. Synthesis of 2- (4-chloro-2- (trifluoromethyl) phenyl) -Y- (6-morpholinopyridin-2-yl) -1H-benzo [D] imidazole-4-carboxamide (compound 868).

Stage 1. Obtaining 6-morpholinopyridin-2-amine (165)

A mixture containing 4-chloro-2-aminopyridine (164; 26 g, 0.20 mol), K ₂ CO ₃ (0.40 mol) and morpholine (48; 0.6 mol) in EM8O (150 ml) was stirred at 190 ° C for 10 hours. After cooling to room temperature, the reaction mixture was diluted with water (300 ml) and the resulting mixture was extracted

- 58 020578 ethyl acetate (4x150 ml). The combined organic layers were washed with water (3x25 ml), dried with AlN (O ₄ ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (petroleum ether: ethyl acetate = 10: 1) to give 6-morpholinopyridin-2-amine 165 (17 g, 47%) as a white solid.

Stage 2. Obtaining 2- (4-chloro-2- (trifluoromethyl) phenyl) -Y- (6-morpholinopyridin-2-yl) -1H-benzo [b] imidazole-4-carboxamide (compound 868)

6-Morpholinopyridin-2-amine 165 was combined with 2- (4-chloro-2- (trifluoromethyl) phenyl) -1Nbenzo [b] imidazole-4-carboxylic acid 54 using the same amide coupling procedure as described above with obtaining 2- (4-chloro-2- (trifluoromethyl) phenyl) -Y- (6-morpholinopyridin-2-yl) 1H-benzo [b] imidazole-4-carboxamide (compound 868). Μδ ^ δΐ) is calculated for С ₂₄ Н1 ₉ С1Р ₃ ЩО ₂ : 501.12; Found: 502 [M + H].

Example 47. Biological activity

Mass spectrometric analysis was used to identify δΙΚΤΙ activity modulators. In the mass spectrometric analysis, a peptide containing 20 amino acid residues was used, such as: Ac-EE-K (biotin) -O ^ δΤδδНδК (Αс) Ν1еδΤΕО-К (5ΤΜΚ) -ΕΕ-ΝН2 (δΙΤ) ΙΌ ΝΘ: 1), where K (Ac) is an acetylated lysine residue and Νΐε is norleucine. The peptide was labeled with 5TMK fluorophore (excitation wavelength 540 nm / emission wavelength 580 nm) at the C-terminus. The sequence of the peptide substrate is based on p53 with some modifications. In addition, the methionine residue present in nature in this sequence has been replaced by norleucine, since methionine can be oxidized during synthesis and purification.

Mass spectrometric analysis was carried out as follows: 0.5 μM peptide substrate and 120 μM βΝΑΌ + were incubated with 10 nM δΙΚΤΙ for 25 min at 25 ° C in the reaction buffer (50 mM TPC acetate, pH 8, 137 mM No. C1, 2 , 7 mM KC1, 1 mM MdS1 _2, 5 mM ΌΤΤ, 0,05% ΒδΑ). Test compounds can be added to the reaction as described above. The δΙΚΤΙ gene was cloned into a vector containing the T7 promoter and transformed into BB21 (EE3). After a 25-minute incubation with δΙΚΤΙ, 10 μl of a 10% formic acid solution was added to stop the reaction. The reaction mixtures were sealed and frozen for further use in mass spectrometric analysis. Determining the mass of the peptide substrate allows you to accurately determine the degree of acetylation (i.e., the starting material) compared to the deacetylated peptide (product).

The inhibition of sirtuin activity was monitored by adding 1 μl of 500 mM nicotinamide as a negative control at the beginning of the reaction (for example, to determine the maximum inhibition of sirtuin). Activation of sirtuin activity was monitored using 10 nM sirtuin protein, with 1 μl ^ ΜδО instead of compound, for quantitative determination of substrate deacetylation at a given point in time within the linear limits of the analysis. This time point is the same as that used for the test compounds, and, within linear limits, the end point represents the change in speed.

For the above analysis, δΙΚΤΙ protein was expressed and purified as follows. The δΙΚΤΙ gene was cloned into a vector containing the T7 promoter and transformed into BB21 (OE3). The protein was expressed by induction using 1 mM ΙΡΤΟ as a fusion protein with a Ν-terminal H15 tag at 18 ° C. overnight and collected at 30000 xd. Cells were lysed with lysozyme in lysis buffer (50 mM tac-HCl, 2 mM tac [2-carboxyethyl] phosphine (TCEP), 10 μM ΖηΟΕ, 200 mM No. C1) and then sonicated for 10 min for complete lysis. The protein was purified using a VI-ΝΤΑ column (Ashegkash), and the fractions containing pure protein were combined, concentrated and passed through a column with a sieve for size separation (BCPNabs δ200 26/60 d1oBa1). A peak containing soluble protein was collected and processed on an ion exchange column (MonoO). Gradient elution (200-500 mm No. C1) gave pure protein. This protein was concentrated and dialyzed against dialysis buffer (20 mM tac-HCl, 2 mM TCEP) overnight. The protein was aliquoted and frozen at -80 ° C until further use.

Sirtuin-modulating compounds that activated δΙΚΤΙ were identified using the assay described above and are presented in the table below. EC ^ values for activating compounds are presented as A (EC1 _{> 5} <1.0 μM), B (EC _{1> 5} 1-25 μM), C (EC _{1> 5} > 25 μM). The percentage of maximally increased activation is presented as A (increased activation> 200%) or B (increased activation <200%).

- 59 020578

- 60,020,578

210 407 Teas ΟΓΊΝΗ ό Τ'- ρ V / Β Β ού- X 211 346 Ο ^ ΝΗ ό Β Α 05 ““ V / SG-ΝΗ 212 339 ό Β Α Ρ LJ-Ν “V / 213 357 SG-ΝΗ Ο Ρ Β Α Η οχ 214 357 sgnn ό FROM Β 0 ~ Ο Ο ^ ΝΗ ό 215 322 Β Α _........ - one." 03- RJ ^R /> Ο ^κ 216 407 Ο ^ ΝΗ ό FROM Β Ρ _ν / \ _X> ο> Ό 217 407 θΑΐΗ ό FROM Β Η οχ Ρ ^ Ζ /> Λχ ' 218 407 ζγ-ΝΗ ό Ρ Β Α C1 ι ^ Τ \ kA / “X / 219 355 SG-ΝΗ ό Β Α γΛ Ρ V / sg-in ό C1 220 373 Β Α

- 61 020578

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620 456 ρ Ο ^ ΝΗ ³ Λ ^ν Ρ-Ο Β Α 821 423 Ρ ^Ρ '^ 4τ-Ν> = \ тт ^ -4 / ^- ° X — ν θΑ Η Ο Α Α 822 437 Ρ ^Ρ Χ- ^ γ- ' ^Ν κ / = \ ΎΧ> ~ ζ X ^е1 <La Α Α 823 417 OS ^ W ^ ^-01 Ο ^ ΝΗ ό Α Α 824 407 Ρ TXRSR Ο- ^ ΝΗ Ν ^ δ Α Α 825 421 Ρ Ύ / C UH g ~ * ζΡ ~~ ΝΗ mAz Ρ Α Α 826 401 Ρ Ύ'χν-νΡ Ο ’^ ΝΗ ό Α Α 327 472 \ = χ SCH ^ CH7 ohm Α ^ ‘- ο'Ύ'-οη ΟΗ Α Α 828 472 South OSKhch Ο ^ ΝΗ ό Α Α 829 473 Ρ ρ— (ΟΗ ^Ρ Ό: νΑ- ^ ^0Η Χ? ^ Ν \ -! Ί Ο ^ ΝΗ ό Α Α 830 473 Ρ— <ΟΗ ^ρ γν ^Ν Ρν Α- »1 Τ -AA d ~ ° \ -ΰ Ο ^ ΝΗ ά Α Α 331 479 Ρ Ρ— / ΟΗ ^P THIH ^<OH γ-Ν ΡΡ Ο ^ ΝΗ ό Α Α

- 119 020578

832 389 Ρ ΪΧοΟ Ο ^ ΝΗ Az \ = / Α Α 833 403 Ρ ρΧ-Ν ° λ ρ Α Α 834 383 Ρ itz) θΧΝ _Η ό Α Α 835 407 / = \ ΤΧθ4 Ο = * ΝΗ ^Ρ Ο Α Α 836 421 Ρ) = \ υχρ Ο ^1β * '“ΝΗ Ι-Χδ 4 Α Α 837 401 Ρ / = l xx> I o ^ = ~~ And ^p ό Α Α 838 466 p Ρ (LC NM'CHE Αν V Α Α 838 479 Ρ Ρ— <OH ο + 'νη ό Α Α 840 473 ρ p— (OH CHKh> Sh ° ^N Ο ^ ΝΗ ύ Α Α 841 473 Ρ ρ-h he νΥν-Χ-Ζν ™ OSH ό Α Α 842 435 ρχο ST-MN Α ^ si Α Α

- 120 020578

843 435 HA ί X. -LF & - 4ρ - Ν ν SHMN • Λ <o BUT BUT 844 435 ίΧν-ο * </ Χ νΧ BUT BUT 845 470 Ρ oh very <4 BUT BUT 846 488 Ρ 0X99% <ρ ^ _Ν \ __ y (Τ ^ ΝΗ ο-9 BUT BUT 847 449 RFh - L, δ ^ Χ (ί Ί / Н \ Η ΗΜ ^ Ο Α ^Μ Χό BUT BUT 848 455 GGMU ΗΝ ^ Ο BUT. <ο BUT BUT 849 504 Ρ ΟίΑΟ-θ · <ρί ^ _Ν SUMMARY ό O- < BUT BUT 850 415 = Ν ΧΛ-ν cxn Αν <^ ο iho BUT BUT 851 432 Ρ Γ X · <ΧΧ γ4 cxn όοα BUT BUT 852 450 Ρ ΟΧ <ζ O''NN CCA BUT BUT

- 121 020578

- 122 020578

863 446 S a ^r OUCHUS ^ STUN ό Α Α 864 544 ooooh SGUN Wo Α Α 865 446 He _and yyy SGUN 6 Α Α 866 463 ^c h, ΤΎ-Ν> = \ Г ~ \ иУЛУ ^ ° СГУУ Α Α 867 453 ^c h ιΟΆ) = \ r ~ \ 1U Ά Ά ρ '-Υ'Ν' —Ν / SG 'ΝΗ ό Α Α ....... __ 868 502 At GU UU ° ’ οΓίΗ Γν At Α Α 869 548 ί y) —V / - \ -γ'Ν Ν-1 NM ^ O f Ό Α Α 870 479 GU2U GULUU ΙΗνΎ f Α Α 871 472 U-UM, I * —N 1 NG-GU ₅ Χν \ = / Β Α 872 456 yiwu » ί 1 now Υν Μ Α Α

- 123 020578

^Pz % \ L Αχ X 1 ΗΙΤΧ) f 873 548 about BUT BUT

In another embodiment, the compound of the present invention is selected from any of the compounds

№№ 202, 203, 208, 226, 243, 247, 254, 262, 269, 287, 288, 289, 290, 295, 300 302, 304, 312, 313, 315, 316, 317, 319, 321, 323, 327, 328, 340, 343, 344, 345, 346, 354, 356, 357, 358, 359, 360 361, 362, 368, 369, 370, 371, 372, 376, 377, 379, 380, 381, 382, 394, 395, 396, 398, 399, 409, 411, 412, 413, 414, 430, 432, 440, 446, 448, 451, 452, 455, 456, 457, 458, 461, 464, 465, 478, 479, 480 481, 492, 493, 494, 495, 514, 515, 516, 531, 540, 541, 549, 550 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 573, 574, 575, 589, 590, 591, 592, 593, 594, 595, 596, 601, 603, 604, 620, 622, 623, 628, 629, 630, 631, 640, 641, 642, 643, 644, 645, 646, 647, 672, 674, 675, 677, 678, 679, 680, 681, 689, 690, 691, 692, 698, 699, 700 701, 706, 717, 718, 719, 720, 721, 722, 723, 124, 725, 134, 735, 736, 737, 740, 741, 742, 743, 744, 745, 746, 749 151, 7 67, 768, 769, 770, 774, 775, 776, 782, 785, 786, 789, 798, 811, 817, 818, 819, 820, 824, 825, 827, 829, 831, 832, 833, 834, 836, 839, 840, 841, 845, 847, 849, 851, 852, 853, 854, 855, 862, 863, 865, 866, 867 or 868

from the table above.

Equivalents

The present invention provides, inter alia, sirtuin activating compounds and methods for their use. Although specific embodiments of the present invention have been discussed, the above description is illustrative and not limiting. Many variations of the present invention will be apparent to those skilled in the art upon consideration of this description. The full scope of the present invention should be determined by the claims, together with the full scope of equivalents, and description, together with such options.

Link inclusion

All publications and patents referred to in this application, including those listed below, are incorporated into this application by reference in its entirety, as if each individual publication or patent were expressly and individually indicated to be incorporated by reference. In case of conflict, control is the present application, including any definitions contained therein.

Any polynucleotide and polypeptide sequences having a reference to an access number corresponding to an incoming number in a public database, for example, ΤΗε ΙηκΙίΙυΙο £ from Sepotu KezeatsN (ISK) (\ y \ y \ u.Pdg.ogd), are also included by reference in their entirety. and / or 1Hc ΝαΙίοηηΙ с Ш с с Г от от от от от от о оду оду 1 п па па па 1 ((Νί,,, ΒΙ \ \ \ \ \ \ \ \ \ \ \,,,, (\ \ \ \

Claims (15)

ΦΟΡΜΥΠΑ SELECT 1. The compound of formula (II) or its salt, where one of Ζ ¹¹ and Ζ ¹² is SK ¹³ , and the other of Ζ ¹¹ and Ζ ^{12 is} selected from Ν and SK ¹³ , where K is selected from hydrogen, halogen, -OH, -C ^ H fluoro-substituted C ₁ -C ₂ alkyl, -O- (C ₁ -C ₂ fluoro-substituted 124 020578 alkyl), -8- (C ₁ -C ₂ fluoro-substituted alkyl ), C1-C4 alkyl, - (C1-C2 alkyl) -N (R ¹⁴⁾ (R ¹⁴⁾ -OSN2SN (OH) CH2OH, -O- (C1-C4) alkyl, -O4S1-C3) alkyl-N (K ¹⁴ ) (K ¹⁴ ), -Y (K ¹⁴ ) (K ¹⁴ ), -8- (C1-C4> alkyl and C3C ₇ cycloalkyl; K is selected from hydrogen, - (C ₂ -C ₄ ) alkyl, - (C ₁ -C ₄ ) fluoro-substituted alkyl, - (C-CDalkyl-K ⁷ ) ^ ⁷ ), (C1-C4) alkyl-C (O ) -X (K ⁷ ) (K ⁷ ), - (C2-C ₄ ) alkyl-O-K ⁷ and - (C ₂ -C ₄ ) alkyl-K (K ⁷ ) -C (O) -K ⁷ , where each K ^{7 is} independently selected from hydrogen and —C ₁ -C ₄ alkyl; or two K ⁷ bonded to the same nitrogen atom taken together with the nitrogen atom form a 4-7 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (= O) ₂ and O, where the saturated heterocycle is optionally substituted on one carbon atom —OH, —C ₁ -C ₄ alkyl, fluorine, —ΝΗ ₂ , —ΝΗ (Ο ₁ -0 ₄ alkyl), —Y (C ₁ -C ₄ alkyl ) ₂ , -ΝΗ ^ Η ^ Η ^ ΟΗ ^ or -Ы (CH ₂ CH ₂ OCH ₃ ) ₂ ; K ^{4 is} selected from hydrogen, halogen, —C ^ N, fluorinated C ₁ -C ₂ alkyl, -8- (C ₁ -C ₂ ) fluorinated alkyl, C ₁ -C ₄ alkyl, -8- (C ₁ -C ₄ ) alkyl and C ₃ -C ₇ cycloalkyl; X is selected from C (= O) -SK ¹⁵ K ^{1 €} -1 and -SK ¹⁵ K ¹⁶ -HN-C (= O) -Ο-ΐ, where ΐ indicates where X is associated with K ¹¹ K ¹⁵ and K ^{16 are} independently selected from hydrogen, C ₁ -C ₄ alkyl, CP ₃ and - (C ₁ -C ₄ alkyl) -CP ₃ ; K ¹¹ is a heterocycle that is a 4-7 membered monocyclic ring or an 8-12 membered bicyclic ring, as well as a spiro condensed and bridged polycyclic ring system comprising one or more heteroatoms selected from Ν, O and 8 atoms wherein the monocyclic ring and each of the bicyclic rings are independently selected from a saturated, unsaturated or aromatic ring, where K ^{11 is} optionally substituted with one or two substituents independently selected from halogen, ΡξΝ. C1-C3 alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, = O, -O-C1-C4 alkyl, -8-K ¹⁴ , - (C1C4 alkyl) -Y (K ¹⁴ ) (K ¹⁴ ), - N (R ¹⁴⁾ (R ¹⁴⁾ -OTSS2-C4 alkyl) -N (R ¹⁴⁾ (R ^14), -C (O) -N (R ¹⁴⁾ (R ^14), -C (O) -O -K ¹⁴ and - (C1-C4 alkyl) -C (O) -Y (K ¹⁴ ) (K ¹⁴ ), where each K ^{14 is} independently selected from hydrogen and-C ₁ -C ₄ alkyl; or two K ¹⁴ taken together with the nitrogen atom to which they are attached form a 4-8 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, 8, 8 (= O), 8 (O) 2 and O where, when K ¹⁴ is alkyl, such alkyl is optionally substituted with one or more groups —OH, —O— (C ₁ -C ₄ alkyl), fluoro, —, ₂ , —NI (C ₁ -C ₄ alkyl), - S (C ₁ -C ₄ alkyl) ₂ , -ΚΗ ^ Η ^ Η ^^^ or -Κ ^ Η ^ Η ^ ΘΗ ^, and when two К ¹⁴ , taken together with the nitrogen atom to which they are bonded, form 4 An 8-membered saturated heterocycle, such a saturated heterocycle is optionally substituted at the carbon atom by the OH group, -C ₁ -C ₄ alkyl, fluoro, -ΝΗ ₂ , -UI (C1-C ₄ alkyl), -Ы (C ₁ -C ₄ alkyl) ₂ , -ΝΗ ^ Η ^ Η ^ ΩΗ ^ or S (CH ₂ CH ₂ OCH ₃ ) ₂ ; and optionally substituted at any substituted nitrogen atom with a —C ₁ -C ₄ alkyl group, a fluoro-substituted C ₁ -C ₄ alkyl, or - (CH ₂ ) ₂ —O — CH ₃ ; K ^{12 is} selected from a carbocycle, which is a 5-7 membered monocyclic ring system, independently selected from a saturated, unsaturated and aromatic ring system, where all ring atoms are carbon, and a heterocycle defined for K ¹¹ other than piperazine, indazole , triazole or pyrazolopyridine, wherein K ^{12 is} optionally substituted with one to two substituents independently selected from halogen, —C ^ S, C1-C4 alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, —O — K ¹⁴ , -8- K ¹⁴ , -8 (O) -K ¹⁴ , -8 (O) 2-K ¹⁴ , - (C1-C4 alkyl) -Y (K ¹⁴ ) (K ¹⁴⁾ -N (R ¹⁴⁾ (R ^14), -O _(C2-C4 alkyl) -K (R ¹⁴⁾ (R ^14), -C (O) -N (R ¹⁴⁾ (R ^14), - (C1-C4 alkyl) -C (O) -Y (K ¹⁴ ) (K ¹⁴ ), -O-phenyl, phenyl and the second heterocycle defined for K ¹¹ , and when K ¹² is phenyl, K ^{12 is} also optional substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted with 3,4-ethylenedioxy or -O- (saturated heterocycle), wherein any phenyl or second heterocyclic substituent of the radical K ^{12 is} optionally substituted with halogen; -ί '= Ν; C1-C ₄ alkyl, C ₁ -C ₂ fluoride-substituted alkyl group -O- (C ₁ -C ₂ fluorine-substituted alkyl), -O- (C ₁ -C ₄ alkyl) -8- (C ₁ -C ₄ alkyl ), -8 (C ₁ -C ₂ fluoro-substituted alkyl), -ΝΗ - ^ - ^ alkyl) and -Ы- (C ₁ -C ₄ alkyl) ₂ . - 125 020578 2. The compound according to claim 1, having the formula 3. The compound of formula (Ι) or its salt, where one of Ζ and Ζ is SC, and the other of Ζ and Ζ is selected from Ν and SC, where K ^{3 is} selected in each case from hydrogen, hydroxy, halogen, -ΝΝ, fluoro-substituted C1-C2 alkyl, -O- (O-C ₂ ) fluoro-substituted alkyl, A- (C1-C ₂ ) fluoro-substituted alkyl, C1-C4 alkyl, -O- (O-C ₄ ) alkyl, -8- (C1-C4) alkyl, C3-C7 cycloalkyl; K is selected from hydrogen, - (C ₂ -C ₄ ) alkyl, - (C1-C ₄ ) fluoro-substituted alkyl, - (C1-C4) alkyl-I (K ⁷ ) (K ⁷ ), (SGS4) alkyl-C ( O) -Y (K ⁷ ) (K ⁷ ), - (C2-C ₄ ) alkyl-O-K ⁷ and - (C ₂ -C ₄ ) alkyl-O (K ⁷ ) -C (O) -K ⁷ where each K ^{7 is} independently selected from hydrogen and —C ₁ -C ₄ alkyl; or two K ⁷ bonded to the same nitrogen atom taken together with the nitrogen atom form a 4-7 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, δ, 8 (= O), 8 (= O) ₂ and O, where the saturated heterocycle is optionally substituted on one carbon atom with the group —OH, —C1-C ₄ alkyl, fluoro, -ΝΗ ₂ , -ΝΗ ^ ι-Ο alkyl), -I (C1-C ₄ alkyl) ₂ , ΝΗ ^ Η ^^ Ν) or -Y (CH ₂ CH ₂ OCH ₃ ) ₂ ; K ^{1 is} selected from a carbocycle, which is a 5-7-membered monocyclic or 8-12 membered bicyclic ring system, as well as a spiro-fused and bridged polycyclic ring system, where all ring atoms are carbon, monocyclic or each of bicyclic the rings are independently selected from a saturated, unsaturated and aromatic ring, and a heterocycle, which is a 4-7 membered monocyclic or 8-12 membered bicyclic ring system, as well as a spirocon A polycyclic ring system with a bridged bond comprising one or more heteroatoms selected from Ν, O and δ atoms, the monocyclic ring and each of the bicyclic rings being independently selected from a saturated, unsaturated or aromatic ring, where K ^{1 is} optionally substituted with one or two substituents independently selected from halogen, ΝΝ, O-C3 alkyl, = O, C3-C7 cycloalkyl, fluoro-substituted C1-C2 alkyl, -O-K ⁸ , -δ-K ⁸ , - (C1-C2 alkyl) ^ K ⁸ ) (K ⁸ ), -Y (K ⁸ ) (K ⁸ ), -O- (C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -O- (C- -FROM; alkyl) -Y (K ⁸ ) (K ⁸ ) and - (C1-C2 alkyl) -C (O) -I (K ⁸ ) (K ⁸ ), and when K ¹ is phenyl, K ^{1 is} also optionally substituted 3 4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy and fluoro-substituted 3,4-ethylenedioxy; each K ^{8 is} independently selected from hydrogen and —C 1 -C 4 alkyl; or two K ⁸ taken together with the nitrogen atom to which they are bonded form a 4-8 membered saturated heterocycle, optionally including one additional heteroatom selected from Ν, δ, 8 (= O), 8 (= O) ₂ and O, where the alkyl is optionally substituted with one or more groups —OH, fluoro, —ΝΗ ₂ , ΝΗ (0-0 alkyl), —I (C1-C ₄ alkyl) ₂ , —NΗ (CH ₂ CH ₂ OCH ₃ ), or - S (CH ₂ CH ₂ OCH ₃ ) ₂ , and the saturated heterocycle is optionally substituted at the carbon atom with the group —OH, -C1-C ₄ alkyl, fluoro, -ΝΗ ₂ , -ΝΗ ^ -Ο alkyl), -Y (C1-C ₄ alkyl) ₂ , - ^ ЩСЩСЩСЩСЩ) or -C (CH ₂ CH ₂ ОСН ₃ ) ₂ , K ^{2 is} selected from a carbocycle defined for K ¹ containing at least five ring atoms and a 4-7 membered monocyclic heterocycle defined for K ¹ other than piperazine, where K ^{2 is} optionally substituted with one to two substituents independently selected from halogen, -Ο = Ν, C-C alkyl, C3-C7 cycloalkyl, C1-C2 fluoro-substituted alkyl, -O-K ⁸ , -δ-K ⁸ , - (C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ), ^ K ⁸ ) (K ⁸ ), -O- (C1-C2 alkyl) -Y (K ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -O- (C - C; alkyl) -Y (K ⁸ ) (K ⁸ ), -C (OLC ⁸ ) (K ⁸ ), - (C1-C2 alkyl) -C (O) -Y (K ⁸ ) (K ⁸ ), -O-phenyl, phenyl and second heterocycle as defined for R ^1, where K ² is phenyl, R ² is also optionally substituted 3,4metilendioksi, 3,4metilendioksi fluoride-substituted, 3,4-ethylenedioxy, or fluoride-substituted 3,4-ethylenedioxy, wherein any phenyl or second heterocycle substituent K ² optionally substituted by halogen; -ΝΝ; _O-C3 alkyl, C1-C2 fluoride-substituted alkyl, -O- (C _O-2) fluoride-substituted alkyl, -O- (O-C ₃₎ alkyl -8- (O-C _s) alkyl, A-ui SD fluoride-substituted alkyl, -ΝΗ- ^ ι-Ο) alkyl and -Y- _{(C1-C3) 2} alkyl; and K ^{4 is} selected from hydrogen, halogen, -ΝΝ, fluoro-substituted С1-С2 alkyl, А-Щ-СД fluoro-substituted-126 020578 alkyl, С ₁ -С ₄ alkyl, -8- (С ₁ -С ₄ ) alkyl and С ₃ -C ₇ cycloalkyl; X is selected from -YN-C (= O) - +, -C (= 0) -ΝΗ-1, where 1 indicates where X is bonded to K ¹ ; and each K ⁵ and K ^{6 is} independently selected from hydrogen, C ₁ -C ₄ alkyl, -CP ₃ and (C ₁ -C ₂ alkyl) -CP ₃ , where when X is -C (0) -ΝΗ-Ι, each Ζ ¹ and Ζ ² are CH and K ¹ is optionally substituted phenyl, then K ² is different from pyridinyl. 4. The compound according to claim 3, having the formula 5. The compound according to any one of claims 1 to 4, where K is hydrogen. 6. The compound according to any one of claims 1 to 5, where X is -C (= 0) -ΝΗ-1, 7. The compound according to claim 1 or 2, where K ^{11 is} selected from - 127 020578 8. The compound according to any one of claims 1, 2 or 7, where K ^{12 is} selected from ρ - 128 020578 9. The compound according to claim 3 or 4, where K ^{1 is} selected from 10. The compound according to any one of claims 3, 4 or 9, where K ² selected from - 129 020578 - 130 020578 11. The compound according to claim 1, selected from or its salt. 12. The compound according to claim 3, selected from - 131 020578 or its salt. 13. A pharmaceutical composition comprising a compound according to any one of claims 1-12, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 14. The pharmaceutical composition according to item 13, further comprising another active agent selected from anti-inflammatory drugs, analgesics, antimicrobials, antifungal agents, antibiotics, vitamins, antioxidants and sun-blocking agents, including anthranilates, benzophenones (in particular benzophenone-3 ), camphor derivatives, cinnamates (e.g., octyl methoxy cinnamate), dibenzoylmethanes (e.g., butyl methoxybenzoylmethane), paminobenzoic acid (PABA) and its derivatives and salicylates (e.g., octyl itsilat). 15. A method of treating a subject suffering from or sensitive to insulin resistance, metabolic syndrome, diabetes or its complications, or increasing insulin sensitivity in a subject, comprising administering to the subject in need thereof a composition according to claim 13.